Cross-reactive primers for amplifying the nucleic acids of HIV-1 and HIV-2

ABSTRACT

Compositions, methods and kits for detecting the nucleic acids of HIV-1, HIV-2, or the combination of HIV-1 and HIV-2. Particularly described are oligonucleotides that are useful as hybridization probes and amplification primers, including cross-reacting hybridization probes and cross-reacting amplification primers, for detecting very low levels of viral nucleic acids.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 11/015,605,filed Dec. 16, 2004, now U.S. Pat. No. 7,255,996, which claims thebenefit of U.S. Provisional Application Nos. 60/531,183, filed Dec. 19,2003, and 60/584,706, filed Jun. 30, 2004. The entire disclosures ofthese prior applications are hereby incorporated by reference.

GOVERNMENT INTEREST IN INVENTION

Certain aspects of the invention disclosed herein were made withgovernment support under contracts N01-HB-67130 and N01-HB-07148 withthe National Heart, Lung and Blood Institute of the National Institutesof Health. The United States government has certain rights in theseaspects of the invention.

FIELD OF THE INVENTION

The present invention relates to the subfield of biotechnology concernedwith nucleic acid amplification technology. More specifically, theinvention relates to individual assays that are capable of detecting thenucleic acids of HIV-1, HIV-2, or the combination of HIV-1 and HIV-2,and further relates to multiplex assays that are capable of detectingthe nucleic acids of both HIV-1 and HIV-2 using a probe and/or primersthat cross-react with the two analytes.

BACKGROUND OF THE INVENTION

Although the HIV/AIDS pandemic is principally due to infection by HIV-1,a different retrovirus has emerged as another cause of AIDS. Thisso-called “HIV-2” virus was first isolated from AIDS patients in WestAfrica in 1986, and was subsequently detected as an infectious agent forthe first time in the United States the following year. Fewer than 100cases of HIV-2 had been reported in the United States through the end of1994. Despite this seemingly low number, HIV-2 is being identified asthe etiologic agent in growing numbers of immunosuppressive diseasesthat are clinically indistinguishable from AIDS cases that result fromHIV-1 infection (Kanki et al., Science 232:238 (1986); Kanki et al.,Science 236:827 (1987); Clavel et al., Science 233:343 (1986); Clavel etal., N. Engl. J. Med. 316:1180 (1987)). Although HIV-2 is related toHIV-1 by its morphology and tropism for CD4⁺ cells, it clearly is adistinct virus and not merely an envelope variant of HIV-1.

Indeed, since HIV-2 is only distantly related to HIV-1, withapproximately 50% amino acid conservation in the gag and pol proteinsand less than 30% conservation in the env gene products, its presence isnot effectively detected by serologic assays used for detecting HIV-1infection (Constantine N T, AIDS 7:1 (1993); Markovitz D M, Ann. Intern.Med. 118:211 (1993)). As a result, attempts have been made to developnucleic acid probes that can be used for specifically detecting HIV-2viral nucleic acids.

Interestingly, the genomes of both HIV-1 and HIV-2 show substantialsequence heterogeneity among different isolates. As a consequence ofthis heterogeneity, it has been impossible to find substantial regionsof absolute sequence conservation between all isolates of HIV-1 or allisolates of HIV-2 (see published European Patent Application EP 0 887427). Indeed, numerous viral isolates with unique polynucleotidesequences have been identified for each of these viruses, a factor thatfurther complicates the construction of probes for reliable andeffective nucleic acid testing.

Since, like HIV-1, HIV-2 also is transmissible through exchange of bodyfluids, including blood and plasma, it is important to be able to detectinfected body fluids before antibodies to the virus are detectable orsymptoms are evident in an infected individual. For protection ofpatients who might otherwise receive an HIV-2-infected body fluid (e.g.,whole blood or plasma during transfusion), or products derived fromdonated blood or plasma, it is particularly important to detect thepresence of the virus in the donated body fluid to prevent its use insuch procedures or products. It is also important that procedures andreagents used for detecting HIV-2 can detect relatively low numbers ofviral copies which may be present in an infected individual, who may bea donor, during the early stages of infection.

Assays and reagents for detecting HIV-2 have been previously disclosedin, for example, U.S. Pat. Nos. 6,020,123, 5,688,637, 5,545,726 and5,310,651; European Patent Nos. EP 0404625 B1 and EP 0239425 B1; andpublished European Patent Application Nos. EP 1026236 A2, EP 0887427 A2.

SUMMARY OF THE INVENTION

A first aspect of the invention relates to a method for determiningwhether a test sample contains an HIV-1 analyte nucleic acid or an HIV-2analyte nucleic acid. The invented method includes a first step forcombining the test sample with a pair of cross-reactive primers. Next,there is a step for amplifying in an in vitro nucleic acid amplificationreaction any of a first sequence of the HIV-1 analyte nucleic acid thatmay be present in the test sample and any of a first sequence of theHIV-2 analyte nucleic acid that may be present in the test sample. Thisnucleic acid amplification reaction uses a pair of cross-reactiveprimers that are capable of co-amplifying HIV-1 and HIV-2 nucleic acids.Products of the reaction may include a first HIV-1 amplicon and a firstHIV-2 amplicon. Next, there is a step for detecting in a singlehybridization reaction any of the first HIV-1 amplicon and any of thefirst HIV-2 amplicon that may have been synthesized in the amplifyingstep. A positive result in the hybridization reaction will indicate thatthe test sample contained at least one of either the HIV-1 analytenucleic or the HIV-2 analyte nucleic acid. In a preferred embodiment,the in vitro nucleic acid amplification reaction in the amplifying stepis either a TMA reaction, a NASBA reaction or a PCR reaction. In anotherpreferred embodiment, the single hybridization reaction in the detectingstep involves the use of a cross-reactive probe that can hybridizeeither to the first HIV-1 amplicon or to the first HIV-2 amplicon. Morepreferably, the hybridization reaction in the detecting step furtherincludes an HIV-1-specific probe which hybridizes only to the firstHIV-1 amplicon and not to the first HIV-2 amplicon. When this is thecase, a positive signal indicating hybridization of the cross-reactiveprobe together with the absence of a positive signal indicatinghybridization of the HIV-1-specific probe indicates that the test samplecontains the HIV-2 analyte nucleic acid and does not contain the HIV-1analyte nucleic acid. In an alternative preferred embodiment, there isan additional step for detecting in a hybridization reaction thatincludes an HIV-1-specific probe, only the first HIV-1 amplicon and notdetecting the first HIV-2 amplicon. In this instance, the HIV-1-specificprobe hybridizes only to the first HIV-1 amplicon and not to the firstHIV-2 amplicon. When this is the case, a positive signal indicatinghybridization of the cross-reactive probe together with the absence of apositive signal indicating hybridization of the HIV-1-specific probeindicates that the test sample contains the HIV-2 analyte nucleic acidand does not contain the HIV-1 analyte nucleic acid. In accordance withanother preferred embodiment, the cross-reactive probe used in thedetecting step is labeled with a homogeneously detectable label. Thehomogeneously detectable label can be, for example, a chemiluminescentlabel. In a highly preferred embodiment, when a chemiluminescent labelis employed, the detecting step involves detecting with a luminometer,or performing luminometry. In certain other embodiments of the inventedmethod, the in vitro nucleic acid amplification reaction conducted inthe amplifying step does not include an analyte-specific pair of primersthat amplify the first sequence of the HIV-2 analyte nucleic acidwithout also being capable of amplifying the first sequence of the HIV-1analyte nucleic acid. In still other embodiments, a positive resultindicating probe hybridization in the detecting step does notdistinguish between the presence of the first HIV-1 amplicon and thefirst HIV-2 amplicon. Stated differently, hybridization of the probe toa complementary target nucleic acid synthesized in the amplificationreaction indicates only that HIV-1 or HIV-2 nucleic acids were presentin the test sample, without identifying which was present. In yet otherembodiments, the in vitro nucleic acid amplification reaction in theamplifying step can further amplify at least a first sequence of atleast one analyte nucleic acid which is different from HIV-1 and HIV-2.In this instance the amplification reaction would be a “multiplex”amplification reaction. In a particularly preferred embodiment of theinvented method, the nucleic acids of at least one of hepatitis B virusand hepatitis C virus can be amplified in the amplification reaction inaddition to HIV-1 and HIV-2 nucleic acids.

A second aspect of the invention relates to a method for particularlydetermining whether a test sample contains an HIV-1 analyte nucleicacid. The invented method includes a first step for combining the testsample with a pair of cross-reactive primers. Next, there is a step foramplifying in an in vitro nucleic acid amplification reaction any of afirst sequence of the HIV-1 analyte nucleic acid that may be present inthe test sample, and any of a first sequence of an HIV-2 analyte nucleicacid that may be present in the test sample. This amplification reactionis carried out using a pair of cross-reactive primers that are capableof co-amplifying HIV-1 and HIV-2 nucleic acids. Products of theamplification reaction may include a first HIV-1 amplicon and a firstHIV-2 amplicon. Next, there is a step for detecting any of the firstHIV-1 amplicon that may have been synthesized in the amplifying stepwithout detecting any of the first HIV-2 amplicon. A positive result inthe hybridization reaction will indicate that the test sample containedthe HIV-1 analyte nucleic. In a preferred embodiment, the in vitronucleic acid amplification reaction in the amplifying step is either aTMA reaction, a NASBA reaction or a PCR reaction. When one of theseamplification reactions is employed, the detecting step preferablyinvolves hybridizing an HIV-1 specific hybridization probe which islabeled with a homogeneously detectable label. Such labelsadvantageously do not require physical separation of unhybridized, freeprobe from specific probe:target duplexes to determine that suchduplexes have formed in a hybridization reaction. In certain preferredembodiments, the homogeneously detectable label is a chemiluminescentlabel. When this is the case, the detecting step may involve detectingwith a luminometer, or performing luminometry. In still anotherembodiment, the in vitro nucleic acid amplification reaction in theamplifying step does not include an analyte-specific pair of primersthat amplify the first sequence of the HIV-2 analyte nucleic acidwithout also being capable of amplifying the first sequence of the HIV-1analyte nucleic acid. In yet other embodiments, the in vitro nucleicacid amplification reaction in the amplifying step can further amplifyat least a first sequence of at least one analyte nucleic acid which isdifferent from HIV-1 and HIV-2. In this instance the amplificationreaction would be a “multiplex” amplification reaction. For example, ina particularly preferred embodiment of the invented method the nucleicacids of at least one of hepatitis B virus and hepatitis C virus can beamplified in the amplification reaction in addition to HIV-1 and HIV-2nucleic acids.

A third aspect of the invention relates to a method for particularlydetermining whether a test sample contains an HIV-2 analyte nucleicacid. The invented method includes a first step for combining the testsample with a pair of cross-reactive primers. Next, there is a step foramplifying in an in vitro nucleic acid amplification reaction any of afirst sequence of the HIV-1 analyte nucleic acid that may be present inthe test sample, and any of a first sequence of an HIV-2 analyte nucleicacid that may be present in the test sample. This amplification reactionis carried out using a pair of cross-reactive primers that are capableof co-amplifying HIV-1 and HIV-2 nucleic acids. Products of theamplification reaction may include a first HIV-1 amplicon and a firstHIV-2 amplicon. Next, there is a step for detecting any of the firstHIV-2 amplicon that may have been synthesized in the amplifying stepwithout detecting any of the first HIV-1 amplicon. A positive result inthe hybridization reaction will indicate that the test sample containedthe HIV-2 analyte nucleic. In a preferred embodiment, the in vitronucleic acid amplification reaction in the amplifying step is either aTMA reaction, a NASBA reaction or a PCR reaction. When one of theseamplification reactions is employed, the detecting step preferablyinvolves hybridizing an HIV-2 specific hybridization probe which islabeled with a homogeneously detectable label. Such labelsadvantageously do not require physical separation of unhybridized, freeprobe from specific probe:target duplexes to determine that suchduplexes have formed in a hybridization reaction. In certain preferredembodiments, the homogeneously detectable label is a chemiluminescentlabel. When this is the case, the detecting step may involve detectingwith a luminometer, or performing luminometry. In still anotherembodiment, the in vitro nucleic acid amplification reaction in theamplifying step does not include an analyte-specific pair of primersthat amplify the first sequence of the HIV-2 analyte nucleic acidwithout also being capable of amplifying the first sequence of the HIV-1analyte nucleic acid. In yet other embodiments, the in vitro nucleicacid amplification reaction in the amplifying step can further amplifyat least a first sequence of at least one analyte nucleic acid which isdifferent from HIV-1 and HIV-2. In this instance the amplificationreaction would be a “multiplex” amplification reaction. For example, ina particularly preferred embodiment of the invented method the nucleicacids of at least one of hepatitis B virus and hepatitis C virus can beamplified in the amplification reaction in addition to HIV-1 and HIV-2nucleic acids.

A fourth aspect of the invention relates to a method of determiningwhether a test sample contains an HIV-1 analyte nucleic acid. Theinvented method involves first amplifying in a first in vitro nucleicacid amplification reaction any of a first sequence of the HIV-1 analytenucleic acid that may be present in the test sample, and any of a firstsequence of an HIV-2 analyte nucleic acid that may be present in thetest sample. The first amplification reaction uses a pair ofcross-reactive primers that are capable of co-amplifying HIV-1 and HIV-2nucleic acids. Products of the first amplification reaction may includea first HIV-1 amplicon and a first HIV-2 amplicon. Next, there is a stepfor detecting in a single hybridization reaction any of the first HIV-1amplicon and any of the first HIV-2 amplicon that may have beensynthesized in the first amplification reaction. Detection of one of theamplicon species confirms that the test sample contains either HIV-1 orHIV-2 nucleic acids. There is next a step for amplifying in a second invitro nucleic acid amplification reaction any of a second sequence ofthe HIV-1 analyte nucleic acid that maybe present in the test sample,thereby resulting in the synthesis of a second HIV-1 amplicon. Finally,there is a step for detecting the second HIV-1 amplicon using a probethat hybridizes to the second HIV-1 amplicon but not to any HIV-2amplicon that may have been synthesized in the second amplifying step.Detection of the second HIV-1 amplicon will confirm that the test samplecontains the HIV-1 analyte nucleic acid. In a preferred embodiment, thein vitro nucleic acid amplification reaction in the first amplifyingstep is either a TMA reaction, a NASBA reaction or a PCR reaction. Whenthis is the case, the in vitro nucleic acid amplification reaction inthe second amplifying step also can be either a TMA reaction, a NASBAreaction or a PCR reaction. In an alternative embodiment, the in vitronucleic acid amplification reaction in the second amplifying step iseither a TMA reaction, a NASBA reaction or a PCR reaction, regardless ofthe type of amplification reaction employed in the first amplifyingstep. In accordance with a different preferred embodiment, the first invitro nucleic acid amplification reaction and the second in vitronucleic acid amplification reaction employ different primers tosynthesize the first HIV-1 amplicon and the second HIV-1 amplicon. Inaccordance with still a different preferred embodiment, the first andsecond detecting steps do not employ identical probes. However, it ispreferred for the single hybridization reaction of the first amplifyingstep to include a cross-reactive probe which is capable of hybridizingeither to the first HIV-1 amplicon or to the first HIV-2 amplicon. Stillmore preferably, the cross-reactive probe is labeled with ahomogeneously detectable label. In certain embodiments, thehomogeneously detectable label is, for example, a chemiluminescentlabel.

A fifth aspect of the invention relates to a method of amplifying anHIV-1 analyte nucleic acid and an HIV-2 analyte nucleic acid that may bepresent in a test sample. The invented method begins with a step forcombining the test sample with a pair of cross-reactive primers. Theseprimers include a cross-reactive first primer that independentlyhybridizes to any of a first strand of the HIV-1 analyte nucleic acidand any of a first strand of the HIV-2 analyte nucleic acid, if presentin the test sample. Also included in the pair of cross-reactive primersis a cross-reactive second primer that independently hybridizes to anyof a second strand of the HIV-1 analyte nucleic acid and any of a secondstrand of the HIV-2 analyte nucleic acid, if present in the test sample.The primers have sequences such that an extension product of thecross-reactive first primer, using as a template either the first strandof the HIV-1 analyte nucleic acid or the first strand of the HIV-2analyte nucleic acid, hybridizes to the cross-reactive second primer.Next, there is a step for amplifying in an in vitro nucleic acidamplification reaction any of a first sequence of the HIV-1 analytenucleic acid that may be present in the test sample and any of a firstsequence of the HIV-2 analyte nucleic acid that may be present in thetest sample using the pair of cross-reactive primers. This results in afirst HIV-1 amplicon being synthesized if the test sample contains theHIV-1 analyte nucleic acid, and a first HIV-2 amplicon being synthesizedif the test sample contains the HIV-2 analyte nucleic acid. In oneembodiment, the invented method further includes a step for detecting atleast one of the first HIV-1 amplicon and the first HIV-2 amplicon. In apreferred embodiment, the detecting step involves detecting both thefirst HIV-1 amplicon and the first HIV-2 amplicon. More preferably, thedetecting step involves performing a hybridization reaction thatincludes a cross-reactive probe that hybridizes independently to any ofthe first HIV-1 amplicon and any of the first HIV-2 amplicon synthesizedin the amplifying step. In another preferred embodiment, the detectingstep involves detecting only the first HIV-1 amplicon and not detectingthe first HIV-2 amplicon. In still another preferred embodiment, thedetecting step involves detecting only the first HIV-2 amplicon and notdetecting the first HIV-1 amplicon. In accordance with another preferredembodiment, when the invented method further includes a step fordetecting at least one of the first HIV-1 amplicon and the first HIV-2amplicon, it is preferred that either (a) the first sequence of theHIV-1 analyte nucleic acid is contained within the HIV-1 p31 integrasegene and the first sequence of the HIV-2 analyte nucleic acid iscontained within the HIV-2 p31 integrase gene, or (b) the first sequenceof the HIV-1 analyte nucleic acid is contained within the HIV-1 p51reverse transcriptase gene and the first sequence of the HIV-2 analytenucleic acid is contained within the HIV-2 p51 reverse transcriptasegene.

A sixth aspect of the invention relates to a composition for amplifyingany of an HIV-1 or any of an HIV-2 analyte nucleic acid that may bepresent in a biological sample. The invented composition includes across-reactive first primer that independently hybridizes to any of afirst strand of the HIV-1 analyte nucleic acid or any of a first strandof the HIV-2 analyte nucleic acid, if present in the biological sample.Also included in the invented composition is a cross-reactive secondprimer that independently hybridizes to any of a second strand of theHIV-1 analyte nucleic acid and any of a second strand of the HIV-2analyte nucleic acid, if present in the biological sample. Thecross-reactive nature of the primers means that an extension product ofthe cross-reactive first primer, as may be mediated by the activity of atemplate-dependent DNA polymerase using the first strand of either ofthe HIV-1 or HIV-2 analyte nucleic acid as a template, is able tohybridize to the cross-reactive second primer. In a preferredembodiment, the HIV-1 and HIV-2 analyte nucleic acids that can beamplified by the cross-reactive first and second primers encode eitherthe viral p31 integrase of the viral p51 reverse transcriptase. Certainmore preferred embodiments of the invented composition do not include apair of HIV-2-specific primers for amplifying only the HIV-2 analytenucleic acid without also being able to amplify the HIV-1 analytenucleic acid, but may include a pair of HIV-1-specific primers foramplifying only the HIV-1 analyte nucleic acid without also amplifyingthe HIV-2 analyte nucleic acid. In accordance with still anotherembodiment, regardless of whether the cross-reactive first and secondprimers are useful for amplifying nucleic acids encoding the p31integrase or the p51 reverse transcriptase, the invented composition mayfurther include a pair of HIV-1-specific primers for amplifying only theHIV-1 analyte nucleic acid without also amplifying the HIV-2 analytenucleic acid. Generally speaking, when the cross-reactive first andsecond primers are useful for amplifying nucleic acids encoding the p51reverse transcriptase, the cross-reactive first primer includes a 3′terminal target-complementary sequence and optionally a cross-reactivefirst primer upstream sequence that is not complementary to the analytenucleic acid sequence to be amplified. The 3′ tenninaltarget-complementary sequence 30 of the cross-reactive first primerincludes 22-28 contiguous bases contained within SEQ ID NO:60, allowingfor the presence of RNA and DNA equivalent bases and nucleotide analogs.The invented composition further includes a cross-reactive second primerthat includes a 3′ terminal target-complementary sequence and optionallya cross-reactive second primer upstream sequence that is notcomplementary to the target nucleic acid sequence to be amplified. The3′ terminal target-complementary sequence of the cross-reactive secondprimer includes SEQ ID NO:61, allowing for the presence of RNA and DNAequivalent bases and nueleotide analogs. More preferably, the the 3′terminal target-complementary sequence of the cross-reactive firstprimer consists of 22-28 contiguous bases contained within SEQ ID NO:60,allowing for the presence of RNA and DNA equivalent bases and nucleotideanalogs, and the 3′ terminal target-complementary sequence of thecross-reactive second primer consists of SEQ ID NO:61, allowing for thepresence of RNA and DNA equivalent bases and nucleotide analogs. Incertain preferred embodiments, the first primer and the second primerare each up to 60 bases in length. In certain other preferredembodiments, the first primer does not include the optional first primerupstream sequence, the first primer being up to 28 bases in length, andthe second primer is up to 60 bases in length. In still other preferredembodiments, the first primer is up to 60 bases in length, and thesecond primer does not include the optional second primer upstreamsequence, the second primer being 26 bases in length. In yet still otherpreferred embodiments, the first primer does not include the optionalfirst primer upstream sequence, the first primer being up to 28 bases inlength, and the second primer does not include the optional secondprimer upstream sequence, the second primer being 26 bases in length.When this is the case, meaning that the first primer is up to 28 basesin length and the second primer is 26 bases in length, there are certainpreferred combinations of primers that can be used in the inventedcombination. In a first preferred combination, the 3′ terminaltarget-complementary sequence of the first primer is SEQ ID NO:51, andthe 3′ terminal target-complementary sequence of the second primer isany of SEQ ID NO:47, SEQ ID NO:49 and SEQ ID NO:50. In a secondpreferred combination, the 3′ terminal target-complementary sequence ofthe first primer is SEQ ID NO:52, and the 3′ terminaltarget-complementary sequence of the second primer is SEQ ID NO:48. In athird preferred combination, the 3′ terminal target-complementarysequence of the first primer is SEQ ID NO:53 and the 3′ terminaltarget-complementary sequence of the second primer is any of SEQ IDNO:47, SEQ ID NO:49 and SEQ ID NO:50. In a fourth preferred combination,the 3′ terminal target-complementary sequence of the first primer is SEQID NO:54, and the 3′ terminal target-complementary sequence of thesecond primer is SEQ ID NO:48. In a different embodiment, when the firstprimer and the second primer are each up to 60 bases in length, the 3′terminal target-complementary sequence of the first primer is any of SEQID Nos:51-54. More preferrably, the 3′ terminal target-complementarysequence of the second primer is any of SEQ ID Nos:47-50. In still adifferent preferred embodiment, when the first primer is up to 60 basesin length, and when the second primer does not include the optionalsecond primer upstream sequence, the second primer being 26 bases inlength, the 3′ terminal target-complementary sequence of the secondprimer is any of SEQ ID Nos:47-50. More preferably, the first primerincludes the optional first primer upstream sequence, and the 3′terminal target-complementary sequence of the first primer is any of SEQID NO:51-54. When this is the case, there are certain preferredcombinations of primers that can be used in the invented combination. Ina first preferred combination, the 3′ terminal target-complementarysequence of the first primer is SEQ ID NO:51, and the 3′ terminaltarget-complementary sequence of the second primer is any of SEQ IDNO:47, SEQ ID NO:49 and SEQ ID NO:50. In a second preferred combination,the 3′ terminal target-complementary sequence of the first primer is SEQID NO:52, and the 3′ terminal target-complementary sequence of thesecond primer is SEQ ID NO:48. In a third preferred combination, the 3′terminal target-complementary sequence of the first primer is SEQ IDNO:53, and the 3′ terminal target-complementary sequence of the secondprimer is any of SEQ ID NO:47, SEQ ID NO:49 and SEQ ID NO:50. In afourth preferred combination, the 3′ terminal target-complementarysequence of the first primer is SEQ ID NO:54, and the 3′ terminaltarget-complementary sequence of the second primer is SEQ ID NO:48.Again generally speaking, when the cross-reactive first and secondprimers are useful for amplifying nucleic acids encoding the p31integrase, the cross-reactive first primer includes a 3′ terminaltarget-complementary sequence and optionally a cross-reactive firstprimer upstream sequence that is not complementary to the analytenucleic acid sequence to be amplified. The 3′ terminaltarget-complementary sequence of the cross-reactive first primerconsists of any of SEQ ID NOs: 13-15. The invented composition furtherincludes a cross-reactive second primer that includes a 3′ terminaltarget-complementary sequence and optionally a cross-reactive secondprimer upstream sequence that is not complementary to the target nucleicacid sequence to be amplified. The 3′ terminal target-complementarysequence of the cross-reactive second primer includes the sequenceACARYAGTACWAATGGC (SEQ ID NO:10), allowing for the substitution of up totwo base analogs. In a preferred embodiment, the cross-reactive firstprimer and the cross-reactive second primer are each up to 75 bases inlength. More preferably, the 3′ terminal target-complementary sequenceof the cross-reactive second primer is any of SEQ ID NO:2, SEQ ID NO:7,SEQ ID NO:8 and SEQ ID NO:9. Still more preferably, the cross-reactivefirst primer is SEQ ID NO:14, and the cross-reactive second primer isany of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9. Inaccordance with yet more preferred embodiments, either thecross-reactive first primer is SEQ ID NO:14, and the cross-reactivesecond primer is SEQ ID NO:7, or the cross-reactive first primer is SEQID NO:14, and the cross-reactive second primer is SEQ ID NO:2.

A seventh aspect of the invention relates to a probe for detecting anHIV-1 or an HIV-2 nucleic acid. The invented probe includes a probesequence that consists of a target-complementary sequence of bases, andoptionally one or more base sequences that are not complementary to thenucleic acids that are to be detected. The target-complementary sequenceof bases can be of any of: (a) SEQ ID NO:42 or the complement thereof,allowing for the presence of RNA and DNA equivalent bases; (b) SEQ IDNO:43 or the complement thereof, allowing for the presence of RNA andDNA equivalent bases; or (c) SEQ ID NO:44 or the complement thereof,allowing for the presence of RNA and DNA equivalent bases. In allinstances the hybridization probe has a length of up to 60 bases. In apreferred embodiment, the probe further includes a detectable label. Forexample, the detectable label can be a chemiluminescent label. Inaccordance with a different embodiment, the length of the hybridizationprobe is up to 26 bases. In accordance with still a differentembodiment, the probe does not include the optional one or more basesequences that are not complementary to the nucleic acids that are to bedetected, and the probe sequence is any of SEQ ID NO:42 or thecomplement thereof, SEQ ID NO:43 or the complement thereof, and SEQ IDNO:44 or the complement thereof.

An eighth aspect of the invention relates to another probe for detectingan HIV-1 or an HIV-2 nucleic acid. The invented probe includes a probesequence that consists of a target-complementary sequence of bases, andoptionally one or more base sequences that are not complementary to thenucleic acid that is to be detected. The target-complementary sequenceof bases may be any of SEQ ID Nos:23-36.

DEFINITIONS

The following terms have the following meanings for the purpose of thisdisclosure, unless expressly stated to the contrary herein.

As used herein, a “biological sample” is any tissue orpolynucleotide-containing material obtained from a human, animal orenvironmental sample. Biological samples in accordance with theinvention include peripheral blood, plasma, serum or other body fluid,bone marrow or other organ, biopsy tissues or other materials ofbiological origin. A biological sample may be treated to disrupt tissueor cell structure, thereby releasing intracellular components into asolution which may contain enzymes, buffers, salts, detergents and thelike.

As used herein, “polynucleotide” means either RNA or DNA, along with anysynthetic nucleotide analogs or other molecules that may be present inthe sequence and that do not prevent hybridization of the polynucleotidewith a second molecule having a complementary sequence.

As used herein, a “detectable label” is a chemical species that can bedetected or can lead to a detectable response. Detectable labels inaccordance with the invention can be linked to polynucleotide probeseither directly or indirectly, and include radioisotopes, enzymes,haptens, chromophores such as dyes or particles that impart a detectablecolor (e.g., latex beads or metal particles), luminescent compounds(e.g., bioluminescent, phosphorescent or chemiluminescent moieties) andfluorescent compounds.

A “homogeneous detectable label” refers to a label that can be detectedin a homogeneous fashion by determining whether the label is on a probehybridized to a target sequence. That is, homogeneous detectable labelscan be detected without physically removing hybridized from unhybridizedforms of the label or labeled probe. Homogeneous detectable labels arepreferred when using labeled probes for detecting either HIV-1 or HIV-2nucleic acids. Examples of homogeneous labels include fluorescentlabels, such as those associated with molecular beacons, andchemiluminescent labels such as those detailed by Arnold et al., U.S.Pat. No. 5,283,174; Woodhead et al., U.S. Pat. No. 5,656,207; and Nelsonet al., U.S. Pat. No. 5,658,737. Preferred labels for use in homogenousassays include chemiluminescent compounds (e.g., see Woodhead et al.,U.S. Pat. No. 5,656,207; Nelson et al., U.S. Pat. No. 5,658,737; andArnold, Jr., et al., U.S. Pat. No. 5,639,604). Preferredchemiluminescent labels are acridinium ester (“AE”) compounds, such asstandard AE or derivatives thereof (e.g., naphthyl-AE, ortho-AE, 1- or3-methyl-AE, 2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE,ortho-dimethyl-AE, meta-dimethyl-AE, ortho-methoxy-AE,ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE, ortho-fluoro-AE, 1- or3-methyl-ortho-fluoro-AE, 1- or 3-methyl-meta-difluoro-AE, and2-methyl-AE).

A “homogeneous assay” refers to a detection procedure that does notrequire physical separation of hybridized probe from non-hybridizedprobe prior to determining the extent of specific probe hybridization.Exemplary homogeneous assays, such as those described herein, can employmolecular beacons or other self-reporting probes which emit fluorescentsignals when hybridized to an appropriate target, chemiluminescentacridinium ester labels which can be selectively destroyed by chemicalmeans unless present in a hybrid duplex, and other homogeneouslydetectable labels that will be familiar to those having an ordinarylevel of skill in the art.

As used herein, “amplification” refers to an in vitro procedure forobtaining multiple copies of a target nucleic acid sequence, itscomplement or fragments thereof.

By “target nucleic acid” or “target” is meant a nucleic acid containinga target nucleic acid sequence. In general, a target nucleic acidsequence that is to be amplified will be positioned between twooppositely disposed primers, and will include the portion of the targetnucleic acid that is fully complementary to each of the primers.

By “target nucleic acid sequence” or “target sequence” or “targetregion” is meant a specific deoxyribonucleotide or ribonucleotidesequence comprising all or part of the nucleotide sequence of asingle-stranded nucleic acid molecule, and the deoxyribonucleotide orribonucleotide sequence complementary thereto.

By “transcription associated amplification” is meant any type of nucleicacid amplification that uses an RNA polymerase to produce multiple RNAtranscripts from a nucleic acid template. One example of a transcriptionassociated amplification method, called “Transcription MediatedAmplification” (TMA), generally employs an RNA polymerase, a DNApolymerase, deoxyribonucleoside triphosphates, ribonucleosidetriphosphates, and a promoter-template complementary oligonucleotide,and optionally may include one or more analogous oligonucleotides.Variations of TMA are well known in the art as disclosed in detail inBurg et al., U.S. Pat. No. 5,437,990; Kacian et al., U.S. Pat. Nos.5,399,491 and 5,554,516; Kacian et al., PCT No. WO 93/22461; Gingeras etal., PCT No. WO 88/01302; Gingeras et al., PCT No. WO 88/10315; Malek etal., U.S. Pat. No. 5,130,238; Urdea et al., U.S. Pat. Nos. 4,868,105 and5,124,246; McDonough et al., PCT No. WO 94/03472; and Ryder et al., PCTNo. WO 95/03430. The methods of Kacian et al. are preferred forconducting nucleic acid amplification procedures of the type disclosedherein.

As used herein, an “oligonucleotide” or “oligomer” is a polymeric chainof at least two, generally between about five and about 100, chemicalsubunits, each subunit comprising a nucleotide base moiety, a sugarmoiety, and a linking moiety that joins the subunits in a linear spacialconfiguration. Common nucleotide base moieties are guanine (G), adenine(A), cytosine (C), thymine (T) and uracil (U), although other rare ormodified nucleotide bases able to hydrogen bond are well known to thoseskilled in the art. Oligonucleotides may optionally include analogs ofany of the sugar moieties, the base moieties, and the backboneconstituents. Preferred oligonucleotides of the present invention fallin a size range of about 10 to about 100 residues. Oligonucleotides maybe purified from naturally occurring sources, but preferably aresynthesized using any of a variety of well known enzymatic or chemicalmethods.

As used herein, a “probe” is an oligonucleotide that hybridizesspecifically to a target sequence in a nucleic acid, preferably in anamplified nucleic acid, under conditions that promote hybridization, toform a detectable hybrid. A probe optionally may contain a detectablemoiety which either may be attached to the end(s) of the probe or may beinternal. The nucleotides of the probe which combine with the targetpolynucleotide need not be strictly contiguous, as may be the case witha detectable moiety internal to the sequence of the probe. Detection mayeither be direct (i.e., resulting from a probe hybridizing directly tothe target sequence or amplified nucleic acid) or indirect (i.e.,resulting from a probe hybridizing to an intermediate molecularstructure that links the probe to the target sequence or amplifiednucleic acid). The “target” of a probe generally refers to a sequencecontained within an amplified nucleic acid sequence which hybridizesspecifically to at least a portion of a probe oligonucleotide usingstandard hydrogen bonding (i.e., base pairing). A probe may comprisetarget-specific sequences and optionally other sequences that arenon-complementary to the target sequence that is to be detected. Thesenon-complementary sequences may comprise a promoter sequence, arestriction endonuclease recognition site, or sequences that contributeto three-dimensional conformation of the probe (e.g., as described inLizardi et al., U.S. Pat. Nos. 5,118,801 and 5,312,728). Sequences thatare “sufficiently complementary” allow stable hybridization of a probeoligonucleotide to a target sequence that is not completelycomplementary to the probe's target-specific sequence.

As used herein, an “amplification primer” is an oligonucleotide thathybridizes to a target nucleic acid, or its complement, and participatesin a nucleic acid amplification reaction. For example, amplificationprimers, or more simply “primers,” may be optionally modifiedoligonucleotides which are capable of hybridizing to a template nucleicacid and may have a 3′ end that can be extended by a DNA polymeraseactivity. In general, a primer will have a downstream sequence capableof hybridizing to a target nucleic acid, and optionally an upstreamsequence that is not complementary to the target nucleic acid. Theoptional upstream sequence may, for example, serve as an RNA polymerasepromoter or contain restriction endonuclease cleavage sites.

As used herein, and with reference to oligonucleotide probes or primers,the term “cross-react” or “cross-reactive” or variants thereof meansthat the probes or primers are not strictly specific for a singlespecies of target polynucleotide. A probe that hybridizes to HIV-1nucleic acids but not to HIV-2 nucleic acids cannot be said to becross-reactive. Conversely, a probe that is able to hybridize to bothHIV-1 and HIV-2 target nucleic acids to form detectable hybridizationcomplexes would be considered “cross-reactive.” Similarly,cross-reactive primers are capable of participating in a nucleic acidamplification reaction using either HIV-1 or HIV-2 nucleic acids astemplates to result in the synthesis of HIV-1 amplicons and HIV-2amplicons.

By “substantially homologous,” “substantially corresponding” or“substantially corresponds” is meant that the subject oligonucleotidehas a base sequence containing an at least 10 contiguous base regionthat is at least 70% homologous, preferably at least 80% homologous,more preferably at least 90% homologous, and most preferably 100%homologous to an at least 10 contiguous base region present in areference base sequence (excluding RNA and DNA equivalents). Thoseskilled in the art will readily appreciate modifications that could bemade to the hybridization assay conditions at various percentages ofhomology to permit hybridization of the oligonucleotide to the targetsequence while preventing unacceptable levels of non-specifichybridization. The degree of similarity is determined by comparing theorder of nucleobases making up the two sequences and does not take intoconsideration other structural differences which may exist between thetwo sequences, provided the structural differences do not preventhydrogen bonding with complementary bases. The degree of homologybetween two sequences can also be expressed in terms of the number ofbase mismatches present in each set of at least 10 contiguous basesbeing compared, which may range from 0-2 base differences.

By “substantially complementary” is meant that the subjectoligonucleotide has a base sequence containing an at least 10 contiguousbase region that is at least 70% complementary, preferably at least 80%complementary, more preferably at least 90% complementary, and mostpreferably 100% complementary to an at least 10 contiguous base regionpresent in a target nucleic acid sequence (excluding RNA and DNAequivalents). (Those skilled in the art will readily appreciatemodifications that could be made to the hybridization assay conditionsat various percentages of complementarity to permit hybridization of theoligonucleotide to the target sequence while preventing unacceptablelevels of non-specific hybridization.) The degree of complementarity isdetermined by comparing the order of nucleobases making up the twosequences and does not take into consideration other structuraldifferences which may exist between the two sequences, provided thestructural differences do not prevent hydrogen bonding withcomplementary bases. The degree of complementarity between two sequencescan also be expressed in terms of the number of base mismatches presentin each set of at least 10 contiguous bases being compared, which mayrange from 0-2 base mismatches.

By “sufficiently complementary” is meant a contiguous nucleic acid basesequence that is capable of hybridizing to another base sequence byhydrogen bonding between a series of complementary bases. Complementarybase sequences may be complementary at each position in the basesequence of an oligonucleotide using standard base pairing (e.g., G:C,A:T or A:U pairing) or may contain one or more residues that are notcomplementary using standard hydrogen bonding (including abasic“nucleotides”), but in which the entire complementary base sequence iscapable of specifically hybridizing with another base sequence underappropriate hybridization conditions. Contiguous bases are preferably atleast about 80%, more preferably at least about 90%, and most preferablyabout 100% complementary to a sequence to which an oligonucleotide isintended to specifically hybridize. Appropriate hybridization conditionsare well known to those skilled in the art, can be predicted readilybased on base sequence composition, or can be determined empirically byusing routine testing (e.g., See Sambrook et al., Molecular Cloning, ALaboratory Manual, 2^(nd) ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989) at §§ 1.90-1.91, 7.37-7.57, 9.47-9.51 and11.47-11.57 particularly at §§ 9.50-9.51, 11.12-11.13, 11.45-11.47 and11.55-11.57).

By “capture oligonucleotide” is meant at least one nucleic acidoligonucleotide that provides means for specifically joining a targetsequence and an immobilized oligonucleotide due to base pairhybridization. A capture oligonucleotide preferably includes two bindingregions: a target sequence-binding region and an immobilizedprobe-binding region, usually contiguous on the same oligonucleotide,although the capture oligonucleotide may include a targetsequence-binding region and an immobilized probe-binding region whichare present on two different oligonucleotides joined together by one ormore linkers. For example, an immobilized probe-binding region may bepresent on a first oligonucleotide, the target sequence-binding regionmay be present on a second oligonucleotide, and the two differentoligonucleotides are joined by hydrogen bonding with a linker that is athird oligonucleotide containing sequences that hybridize specificallyto the sequences of the first and second oligonucleotides.

By “immobilized probe” or “immobilized nucleic acid” is meant a nucleicacid that joins, directly or indirectly, a capture oligonucleotide to animmobilized support. An immobilized probe is an oligonucleotide joinedto a solid support that facilitates separation of bound target sequencefrom unbound material in a sample.

By “separating” or “purifying” is meant that one or more components ofthe biological sample are removed from one or more other components ofthe sample. Sample components include nucleic acids in a generallyaqueous solution phase which may also include materials such asproteins, carbohydrates, lipids and labeled probes. Preferably, theseparating or purifying step removes at least about 70%, more preferablyat least about 90% and, even more preferably, at least about 95% of theother components present in the sample.

By “RNA and DNA equivalents” or “RNA and DNA equivalent bases” is meantmolecules, such as RNA and DNA, having the same complementary base pairhybridization properties. RNA and DNA equivalents have different sugarmoieties (i.e., ribose versus deoxyribose) and may differ by thepresence of uracil in RNA and thymine in DNA. The differences betweenRNA and DNA equivalents do not contribute to differences in homologybecause the equivalents have the same degree of complementarity to aparticular sequence.

By “consisting essentially of” is meant that additional component(s),composition(s) or method step(s) that do not materially change the basicand novel characteristics of the present invention may be included inthe compositions or kits or methods of the present invention. Suchcharacteristics include the ability to selectively detect HIV-1, HIV-2,or the combination of HIV-1 and HIV-2 nucleic acids in biologicalsamples such as whole blood or plasma. Any component(s), composition(s),or method step(s) that have a material effect on the basic and novelcharacteristics of the present invention would fall outside of thisterm.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating the various polynucleotidesthat can be used for detecting a target region within the nucleic acidof HIV-1 or HIV-2 (represented by a thick horizontal line). Positions ofthe following nucleic acids arc shown relative to the target region:“Capture Oligonucleotide” refers to the nucleic acid used to hybridizeto and capture the target nucleic acid prior to amplification, where “T”refers to a tail sequence used to hybridize an immobilizedoligonucleotide having a complementary sequence (not shown); “Non-T7Primer” and “T7 Promoter-Primer” represent two amplification primersused for conducting TMA, where “P” indicates the promoter sequence ofthe T7 promoter-primer; and “Probe” refers to the probe used fordetecting amplified nucleic acid.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are compositions, methods and kits for detecting thenucleic acids of HIV-1, HIV-2, br the combination of HIV-1 and HIV-2 inbiological samples such as blood, serum, plasma or other body fluid ortissue. The probes, primers and methods of the invention can be usedeither in diagnostic applications or for screening donated blood andblood products or other tissues that may contain infectious particles.

Introduction and Overview

The present invention includes compositions (i.e., amplificationoligonucleotides or primers, and probes), methods and kits that areparticularly useful for detecting the nucleic acids of HIV-1, HIV-2, orthe combination of HIV-1 and HIV-2 in a biological sample. To designoligonucleotide sequences appropriate for such uses, known HIV-1 andHIV-2 nucleic acid sequences were first compared to identify candidateregions of the viral genomes that could serve as reagents in adiagnostic assay. As a result of these comparisons, particular sequenceswere selected and tested as targets for detection using the captureoligonucleotides, primers and probes shown schematically in FIG. 1.Portions of sequences containing relatively few variants were chosen asstarting points for designing synthetic oligonucleotides suitable foruse in capture, amplification and detection of amplified sequences.

Based on these analyses, the amplification primer and probe sequencespresented below were designed. Those having an ordinary level of skillin the art will appreciate that any primer sequences specific for HIV-1,HIV-2, or the combination of HIV-1 and HIV-2 targets, with or without aT7 promoter sequence, may be used as primers in the various primer-basedin vitro amplification methods described below. It is also contemplatedthat oligonucleotides having the sequences disclosed herein could servealternative functions in assays for detecting HIV-1 and/or HIV-2 nucleicacids. For example, the capture oligonucleotides disclosed herein couldserve as hybridization probes, the hybridization probes disclosed hereincould be used as amplification primers, and the amplification primersdisclosed herein could be used as hybridization probes in alternativedetection assays.

The amplification primers disclosed herein are particularly contemplatedas components of multiplex amplification reactions wherein severalamplicon species can be produced from an assortment of target-specificprimers. For example, it is contemplated that certain preferred primersdisclosed herein can be used in multiplex amplification reactions thatare capable of amplifying polynucleotides of unrelated viruses withoutsubstantially compromising the sensitivities of those assays. Particularexamples of these unrelated viruses include HCV and HBV.

Useful Amplification Methods

Amplification methods useful in connection with the present inventioninclude: Transcription Mediated Amplification (TMA), Nucleic AcidSequence-Based Amplification (NASBA), the Polymerase Chain Reaction(PCR), Strand Displacement Amplification (SDA), and amplificationmethods using self-replicating polynucleotide molecules and replicationenzymes such as MDV-1 RNA and Q-beta enzyme. Methods for carrying outthese various amplification techniques respectively can be found in U.S.Pat. No. 5,399,491, published European patent application EP 0 525 882,U.S. Pat. No. 4,965,188, U.S. Pat. No. 5,455,166, U.S. Pat. No.5,472,840 and Lizardi et al., BioTechnology 6:1197 (1988). Thedisclosures of these documents which describe how to perform nucleicacid amplification reactions are hereby incorporated by reference.

In a highly preferred embodiment of the invention, analyte nucleic acidsequences are amplified using a TMA protocol. According to thisprotocol, the reverse transcriptase which provides the DNA polymeraseactivity also possesses an endogenous RNase H activity. One of theprimers used in this procedure contains a promoter sequence positionedupstream of a sequence that is complementary to one strand of a targetnucleic acid that is to be amplified. In the first step of theamplification, a promoter-primer hybridizes to the target RNA at adefined site, Reverse transcriptase creates a complementary DNA copy ofthe target RNA by extension from the 3′ end of the promoter-primer.Following interaction of an opposite strand primer with the newlysynthesized DNA strand, a second strand of DNA is synthesized from theend of the primer by reverse transcriptase, thereby creating adouble-stranded DNA molecule. RNA polymerase recognizes the promotersequence in this double-stranded DNA template and initiatestranscription. Each of the newly synthesized RNA amplicons re-enters theTMA process and serves as a template for a new round of replication,thereby leading to an exponential expansion of the RNA amplicon. Sinceeach of the DNA templates can make 100-1000 copies of RNA amplicon, thisexpansion can result in the production of 10 billion amplicons in lessthan one hour. The entire process is autocatalytic and is performed at aconstant temperature.

Structural Features of Primers

As indicated above, a “primer” refers to an optionally modifiedoligonucleotide which is capable of participating in a nucleic acidamplification reaction. Highly preferred primers are capable ofhybridizing to a template nucleic acid and have a 3′ end that can beextended by a DNA polymerase activity. The 5′ region of the primer maybe non-complementary to the target nucleic acid. If the 5′non-complementary region includes a promoter sequence, it is referred toas a “promoter-primer.” Those skilled in the art will appreciate thatany oligonucleotide that can function as a primer (i.e., anoligonucleotide that hybridizes specifically to a target sequence andhas a 3′ end capable of extension by a DNA polymerase activity) can bemodified to include a 5′ promoter sequence, and thus could function as apromoter-primer. Similarly, any promoter-primer can be modified byremoval of, or synthesis without, a promoter sequence and still functionas a primer.

Nucleotide base moieties of primers may be modified (e.g., by theaddition of propyne groups), as long as the modified base moiety retainsthe ability to form a non-covalent association with G, A, C, T or U, andas long as an oligonucleotide comprising at least one modifiednucleotide base moiety or analog is not sterically prevented fromhybridizing with a single-stranded nucleic acid. As indicated below inconnection with the chemical composition of useful probes, thenitrogenous bases of primers in accordance with the invention may beconventional bases (A, G, C, T, U), known analogs thereof (e.g., inosineor “I” having hypoxanthine as its base moiety; see The Biochemistry ofthe Nucleic Acids 5-36, Adams et al., ed., 11^(th) ed., 1992), knownderivatives of purine or pyrimidine bases (e.g., N⁴-methyldeoxygaunosine, deaza- or aza-purines and deaza- or aza-pyrimidines,pyrimidine bases having substituent groups at the 5 or 6 position,purine bases having an altered or a replacement substituent at the 2, 6or 8 positions, 2-amino-6-methylaminopurine, O⁶-methylguanine,4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines (see, Cook,PCT Int'l Pub. No. WO 93/13121) and “abasic” residues where the backboneincludes no nitrogenous base for one or more residues of the polymer(see Arnold et al., U.S. Pat. No. 5,585,481). Common sugar moieties thatcomprise the primer backbone include ribose and deoxyribose, although2′-O-methyl ribose (OMe), halogenated sugars, and other modified sugarmoieties may also be used. Usually, the linking group of the primerbackbone is a phosphorus-containing moiety, most commonly aphosphodiester linkage, although other linkages, such as, for example,phosphorothioates, methylphosphonates, and non-phosphorus-containinglinkages such as peptide-like linkages found in “peptide nucleic acids”(PNA) also are intended for use in the assay disclosed herein.

Useful Probe Labeling Systems and Detectable Moieties

Essentially any labeling and detection system that can be used formonitoring specific nucleic acid hybridization can be used inconjunction with the present invention. Included among the collection ofuseful labels are radiolabels, enzymes, haptens, linkedoligonucleotides, chemiluminescent molecules, fluorescent moieties(either alone or in combination with “quencher” moieties), andredox-active moieties that are amenable to electronic detection methods.Preferred chemiluminescent molecules include acridinium esters of thetype disclosed by Arnold et al., in U.S. Pat. No. 5,283,174 for use inconnection with homogenous protection assays, and of the type disclosedby Woodhead et al., in U.S. Pat. No. 5,656,207 for use in connectionwith assays that quantify multiple targets in a single reaction. Thedisclosures contained in these patent documents are hereby incorporatedby reference. Preferred electronic labeling and detection approaches aredisclosed in U.S. Pat. Nos. 5,591,578 and 5,770,369, and the publishedinternational patent application WO 98/57158, the disclosures of whichare hereby incorporated by reference. Redox active moieties useful aslabels in the present invention include transition metals such as Cd,Mg, Cu, Co, Pd, Zn, Fe and Ru.

Particularly preferred detectable labels for probes in accordance withthe present invention are detectable in homogeneous assay systems (i.e.,where, in a mixture, bound labeled probe exhibits a detectable change,such as stability or differential degradation, compared to unboundlabeled probe). While other homogeneously detectable labels, such asfluorescent labels and electronically detectable labels, are intendedfor use in the practice of the present invention, a preferred label foruse in homogenous assays is a chemiluminescent compound (e.g., asdescribed by Woodhead et al., in U.S. Pat. No. 5,656,207; by Nelson etal., in U.S. Pat. No. 5,658,737; or by Arnold et al., in U.S. Pat. No.5,639,604). Particularly preferred chemiluminescent labels includeacridinium ester (“AE”) compounds, such as standard AE or derivativesthereof, such as naphthyl-AE, ortho-AE, 1- or 3-methyl-AE,2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE, ortho-dimethyl-AE,meta-dimethyl-AE, ortho-methoxy-AE, ortho-methoxy(cinnamyl)-AE,ortho-methyl-AE, ortho-fluoro-AE, 1- or 3-methyl-ortho-fluoro-AE, 1- or3-methyl-meta-difluoro-AE, and 2-methyl-AE.

In some applications, probes exhibiting at least some degree ofself-complementarity are desirable to facilitate detection ofprobe:target duplexes in a test sample without first requiring theremoval of unhybridized probe prior to detection. By way of example,structures referred to as “molecular torches” are designed to includedistinct regions of self-complementarity (coined “the target bindingdomain” and “the target closing domain”) which are connected by ajoining region and which hybridize to one another under predeterminedhybridization assay conditions. When exposed to denaturing conditions,the two complementary regions (which may be fully or partiallycomplementary) of the molecular torch melt, leaving the target bindingdomain available for hybridization to a target sequence when thepredetermined hybridization assay conditions are restored. Moleculartorches are designed so that the target binding domain favorshybridization to the target sequence over the target closing domain. Thetarget binding domain and the target closing domain of a molecular torchinclude interacting labels (e.g., fluorescent/quencher) positioned sothat a different signal is produced when the molecular torch isself-hybridized as opposed to when the molecular torch is hybridized toa target nucleic acid, thereby permitting detection of probe:targetduplexes in a test sample in the presence of unhybridized probe having aviable label associated therewith. Molecular torches are fully describedin U.S. Pat. No. 6,361,945, the disclosure of which is herebyincorporated by reference.

Another example of a self-complementary hybridization assay probe thatmay be used in conjunction with the invention is a structure commonlyreferred to as a “molecular beacon.” Molecular beacons comprise nucleicacid molecules having a target complementary sequence, an affinity pair(or nucleic acid arms) holding the probe in a closed conformation in theabsence of a target nucleic acid sequence, and a label pair thatinteracts when the probe is in a closed conformation. Hybridization ofthe target nucleic acid and the target complementary sequence separatesthe members of the affinity pair, thereby shifting the probe to an openconformation. The shift to the open conformation is detectable due toreduced interaction of the label pair, which may be, for example, afluorophore and a quencher (e.g., DABCYL and EDANS). Molecular beaconsare fully described in U.S. Pat. No. 5,925,517, the disclosure of whichis hereby incorporated by reference. Molecular beacons useful fordetecting specific nucleic acid sequences may be created by appending toeither end of one of the probe sequences disclosed herein, a firstnucleic acid arm comprising a fluorophore and a second nucleic acid armcomprising a quencher moiety. In this configuration, the probe sequencedisclosed herein serves as the target-complementary “loop” portion ofthe resulting molecular beacon.

Molecular beacons preferably are labeled with an interactive pair ofdetectable labels. Examples of detectable labels that are preferred asmembers of an interactive pair of labels interact with each other byFRET or non-FRET energy transfer mechanisms. Fluorescence resonanceenergy transfer (FRET) involves the radiationless transmission of energyquanta from the site of absorption to the site of its utilization in themolecule, or system of molecules, by resonance interaction betweenchromophores, over distances considerably greater than interatomicdistances, without conversion to thermal energy, and without the donorand acceptor coming into kinetic collision. The “donor” is the moietythat initially absorbs the energy, and the “acceptor” is the moiety towhich the energy is subsequently transferred. In addition to FRET, thereare at least three other “non-FRET” energy transfer processes by whichexcitation energy can be transferred from a donor to an acceptormolecule.

When two labels are held sufficiently close that energy emitted by onelabel can be received or absorbed by the second label, whether by a FRETor non-FRET mechanism, the two labels are said to be in “energy transferrelationship” with each other. This is the case, for example, when amolecular beacon is maintained in the closed state by formation of astem duplex, and fluorescent emission from a fluorophore attached to onearm of the probe is quenched by a quencher moiety on the opposite arm.

Highly preferred label moieties for molecular beacons include afluorophore and a second moiety having fluorescence quenching properties(i.e., a “quencher”). In this embodiment, the characteristic signal islikely fluorescence of a particular wavelength, but alternatively couldbe a visible light signal. When fluorescence is involved, changes inemission are preferably due to FRET, or to radiative energy transfer ornon-FRET modes. When a molecular beacon having a pair of interactivelabels in the closed state is stimulated by an appropriate frequency oflight, a fluorescent signal is generated at a first level, which may bevery low. When this same probe is in the open state and is stimulated byan appropriate frequency of light, the fluorophore and the quenchermoieties are sufficiently separated from each other that energy transferbetween them is substantially precluded. Under that condition, thequencher moiety is unable to quench the fluorescence from thefluorophore moiety. If the fluorophore is stimulated by light energy ofan appropriate wavelength, a fluorescent signal of a second level,higher than the first level, will be generated. The difference betweenthe two levels of fluorescence is detectable and measurable. Usingfluorophore and quencher moieties in this manner, the molecular beaconis only “on” in the “open” conformation and indicates that the probe isbound to the target by emanating an easily detectable signal. Theconformational state of the probe alters the signal generated from theprobe by regulating the interaction between the label moieties.

Examples of donor/acceptor label pairs that may be used in connectionwith the invention, making no attempt to distinguish FRET from non-FRETpairs, include fluorescein/tetramethylrhodamine, IAEDANS/fluororescein,EDANS/DABCYL, coumarin/DABCYL, fluorescein/fluorescein, BODIPY FL/BODIPYFL, fluorescein/DABCYL, lucifer yellow/DABCYL, BODIPY/DABCYL,eosine/DABCYL, erythrosine/DABCYL, tetramethylrhodamine/DABCYL, TexasRed/DABCYL, CY5/BH1, CY5/BH2, CY3/BH1, CY3/BH2 and fluorescein/QSY7 dye.Those having an ordinary level of skill in the art will understand thatwhen donor and acceptor dyes are different, energy transfer can bedetected by the appearance of sensitized fluorescence of the acceptor orby quenching of donor fluorescence. When the donor and acceptor speciesare the same, energy can be detected by the resulting fluorescencedepolarization. Non-fluorescent acceptors such as DABCYL and the QSY 7dyes advantageously eliminate the potential problem of backgroundfluorescence resulting from direct (i.e., non-sensitized) acceptorexcitation. Preferred fluorophore moieties that can be used as onemember of a donor-acceptor pair include fluorescein, ROX, and the CYdyes (such as CY5). Highly preferred quencher moieties that can be usedas another member of a donor-acceptor pair include DABCYL and the BLACKHOLE QUENCHER moieties which are available from Biosearch Technologies,Inc., (Novato, Calif.).

Synthetic techniques and methods of bonding labels to nucleic acids anddetecting labels are well known in the art (e.g., see Sambrook et al.,Molecular Cloning, A Laboratory Manual, 2^(nd) ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989), Chapter 10; Nelson etal., U.S. Pat. No. 5,658,737; Woodhead et al., U.S. Pat. No. 5,656,207;Hogan et al., U.S. Pat. No. 5,547,842; Arnold et al., U.S. Pat. No.5,283,174; Kourilsky et al., U.S. Pat. No. 4,581,333), and Becker etal., European Patent App. No. 0 747 706.

Chemical Composition of Probes

Probes in accordance with the invention comprise polynucleotides orpolynucleotide analogs and optionally may carry a detectable labelcovalently bonded thereto. Nucleosides or nucleoside analogs of theprobe comprise nitrogenous heterocyclic bases, or base analogs, wherethe nucleosides are linked together, for example by phospohdiester bondsto form a polynucleotide. Accordingly, a probe may comprise conventionalribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA), but also maycomprise chemical analogs of these molecules. The “backbone” of a probemay be made up of a variety of linkages known in the art, including oneor more sugar-phosphodiester linkages, peptide-nucleic acid bonds(sometimes referred to as “peptide nucleic acids” as described byHyldig-Nielsen et al., PCT Int'l Pub. No. WO 95/32305), phosphorothioatelinkages, methylphosphonate linkages or combinations thereof. Sugarmoieties of the probe may be either ribose or deoxyribose, or similarcompounds having known substitutions, such as, for example, 2′-O-methylribose and 2′ halide substitutions (e.g., 2′-F). The nitrogenous basesmay be conventional bases (A, G, C, T, U), known analogs thereof (e.g.,inosine or “I”; see The Biochemistry of the Nucleic Acids 5-36, Adams etal., ed., 11^(th) ed., 1992), known derivatives of purine or pyrimidinebases (e.g., N⁴-methyl deoxygaunosine, deaza- or aza-purines and deaza-or aza-pyrimidines, pyrimidine bases having substituent groups at the 5or 6 position, purine bases having an altered or a replacementsubstituent at the 2, 6 or 8 positions, 2-amino-6-methylaminopurine,O⁶-methylguanine, 4-thio-pyrimidines, 4-amiino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines (see, Cook,PCT Int'l Pub. No. WO 93/13121) and “abasic” residues where the backboneincludes no nitrogenous base for one or more residues of the polymer(see Arnold et al., U.S. Pat. No. 5,585,481). A probe may comprise onlyconventional sugars, bases and linkages found in RNA and DNA, or mayinclude both conventional components and substitutions (e.g.,conventional bases linked via a methoxy backbone, or a nucleic acidincluding conventional bases and one or more base analogs).

While oligonucleotide probes of different lengths and base compositionmay be used for detecting the nucleic acids of HIV-1, HIV-2, or thecombination of HIV-1 and HIV-2, preferred probes in this invention havelengths of up to 100 nucleotides, and more preferably have lengths of upto 60 nucleotides. Preferred length ranges for the inventedoligonucleotides are from 10 to 100 bases in length, or more preferablybetween 15 and 50 bases in length, or still more preferably between 15and 30 bases in length. However, the specific probe sequences describedbelow also may be provided in a nucleic acid cloning vector ortranscript or other longer nucleic acid and still can be used fordetecting target nucleic acids. Thus, useful probes in accordance withthe invention can include a target-complementary sequence of bases whichare of limited length, and one or more appended sequences which are notcomplementary to the target sequence that is to be detected. Forexample, a molecular beacon would include a target-complementary loopsequence flanked by “arm” sequences which are not complementary to thetarget that is to be detected.

Selection of Amplification Primers and Detection Probes

Useful guidelines for designing amplification primers and probes withdesired characteristics are described herein. The optimal sites foramplifying and probing HIV-1 and/or HIV-2 nucleic acids contain two, andpreferably three, conserved regions each greater than about 10-15 basesin length, within about 200-300 bases of contiguous sequence. The degreeof amplification observed with a set of primers or promoter-primersdepends on several factors, including the ability of theoligonucleotides to hybridize to their complementary sequences and theirability to be extended enzymatically. Because the extent and specificityof hybridization reactions are affected by a number of factors,manipulation of those factors will determine the exact sensitivity andspecificity of a particular oligonucleotide, whether perfectlycomplementary to its target or not. The effects of varying assayconditions are known to those skilled in the art, and are described byHogan et al., in U.S. Pat. No. 5,840,488, the disclosure of which ishereby incorporated by reference.

The length of the target nucleic acid sequence and, accordingly, thelength of the primer sequence or probe sequence can be important. Insome cases, there may be several sequences from a particular targetregion, varying in location and length, which will yield primers orprobes having the desired hybridization characteristics. While it ispossible for nucleic acids that are not perfectly complementary tohybridize, the longest stretch of perfectly homologous base sequencewill normally primarily determine hybrid stability.

Amplification primers and probes should be positioned to minimize thestability of the oligonucleotide:nontarget (i.e., nucleic acid withsimilar sequence to target nucleic acid) nucleic acid hybrid. It ispreferred that the amplification primers and detection probes are ableto distinguish between target and non-target sequences. In designingprimers and probes, the differences in these Tm values should be aslarge as possible (e.g., at least 2° C. and preferably 5° C.).

The degree of non-specific extension (primer-dimer or non-targetcopying) can also affect amplification efficiency. For this reason,primers are selected to have low self- or cross-complementarity,particularly at the 3′ ends of the sequence. Long homopolymer tracts andhigh GC content are avoided to reduce spurious primer extension.Commercially available computer software can aid in this aspect of thedesign. Available computer programs include MacDNASIS™ 2.0 (HitachiSoftware Engineering American Ltd.) and OLIGO ver. 6.6 (MolecularBiology Insights; Cascade, Colo.).

Those having an ordinary level of skill in the art will appreciate thathybridization involves the association of two single strands ofcomplementary nucleic acid to form a hydrogen bonded double strand. Itis implicit that if one of the two strands is wholly or partiallyinvolved in a hybrid, then that strand will be less able to participatein formation of a new hybrid. By designing primers and probes so thatsubstantial portions of the sequences of interest are single stranded,the rate and extent of hybridization may be greatly increased. If thetarget is an integrated genomic sequence, then it will naturally occurin a double stranded form (as is the case with the product of thepolymerase chain reaction). These double-stranded targets are naturallyinhibitory to hybridization with a probe and require denaturation priorto the hybridization step.

The rate at which a polynucleotide hybridizes to its target is a measureof the thermal stability of the target secondary structure in the targetbinding region. The standard measurement of hybridization rate is theC₀t_(1/2) which is measured as moles of nucleotide per liter multipliedby seconds. Thus, it is the concentration of probe multiplied by thetime at which 50% of maximal hybridization occurs at that concentration.This value is determined by hybridizing various amounts ofpolynucleotide to a constant amount of target for a fixed time. TheC₀t_(1/2) is found graphically by standard procedures familiar to thosehaving an ordinary level of skill in the art.

Preferred Amplification Primers

Primers useful for conducting amplification reactions can have differentlengths to accommodate the presence of extraneous sequences that do notparticipate in target binding, and that may not substantially affectamplification or detection procedures. For example, promoter-primersuseful for performing amplification reactions in accordance with theinvention have at least a minimal sequence that hybridizes to the targetnucleic acid, and a promoter sequence positioned upstream of thatminimal sequence. However, insertion of sequences between the targetbinding sequence and the promoter sequence could change the length ofthe primer without compromising its utility in the amplificationreaction. Additionally, the lengths of the amplification primers anddetection probes are matters of choice as long as the sequences of theseoligonucleotides conform to the minimal essential requirements forhybridizing the desired complementary sequence.

Tables 1 and 2 present specific examples of oligonucleotide sequencesthat were used as primers for amplifying HIV-1, HIV-2, or thecombination of HIV-1 and HIV-2 nucleic acids in the region encoding p31integrase. Table 1 presents the sequences of primers that weresubstantially complementary to one strand of the different nucleic acidtargets. The illustrative primers presented in Table 1 havetarget-complementary sequences that include a 17-mer core sequence ofACARYAGTACWAATGGC (SEQ ID NO:10) (where “R” represents A/G, and “W”represents A or T/U), allowing for the substitution of up to one, oreven up to two base analogs. Inosine is an example of a highly preferredbase analog that can be used for this purpose, and position 5 and/orposition 11 of the core sequence can be substituted with this baseanalog with very good results. It is preferred for one of the primersused in the amplification procedure to have a target-complementarysequence that contains this 17-mer core. The primer may further includeseveral nucleotides appended to the upstream terminus of the coresequence, and may include a few nucleotides appended to the downstreamterminus of the core sequence. For example, there can be five, or evenmore nucleotides appended to the upstream terminus. It is convenient toinclude one, two, or three nucleotides appended to the downstreamterminus, if desired. Table 2 presents the sequences of thetarget-complementary primers and the full sequences for promoter-primersthat were used during development of the invention. Notably, theoligonucleotide sequences in Table 1 and Table 2 are substantiallycomplementary to opposite strands of the target nucleic acid to beamplified.

Primers useful for amplifying the HIV-1 and/or HIV-2 nucleic acidtargets can include nucleotide analogs. For example, when compared withthe basic primer sequence of SEQ ID NO:5, primers having SEQ ID NO:6,SEQ ID NO:7 and SEQ ID NO:9 differ by the presence of a single inosineresidue at position 16, substitution of a T residue for a C at position10 and an inosine residue at position 16, or inosine substitutions atpositions 10 and 16, respectively. As confirmed by the experimentalfindings presented herein, these base differences conferred beneficialproperties that could not have been predicted in advance of thediscovery described herein. More specifically, the results demonstratedthat one of these mutant primers, when paired with a singleopposite-strand primer, lost specificity for the HIV-1 template andacquired the capacity for amplifying both HIV-1 and HIV-2 templates withsubstantially equal efficiency. This illustrates how certain positionsin the primers may be substituted by modified bases or base analogs.

TABLE 1 Polynucleotide Sequences of Amplification Primers SequenceIdentifier ACAGCAGTACAAATGGCAG SEQ ID NO: 1 ACAACAGTACAAATGGCAGT SEQ IDNO: 2 ACAATAGTACTAATGGCAGT SEQ ID NO: 3 TTAAGACAGCAGTACAAATGGC SEQ IDNO: 4 TAGAGACAGCAGTACAAATGGC SEQ ID NO: 5 TAGAGACAGCAGTACIAATGGC SEQ IDNO: 6 TAGAGACAGTAGTACIAATGGC SEQ ID NO: 7 TAGAGACAGCAGTACTAATGGC SEQ IDNO: 8 TAGAGACAGIAGTACIAATGGC SEQ ID NO: 9

Table 2 presents target-complementary oligonucleotide sequences and thecorresponding promoter-primer sequences that were used for amplifyingHIV-1 and HIV-2 nucleic acid sequences. As indicated above, allpromoter-primers included sequences that were substantiallycomplementary to, meaning that they were able to hybridize to, a targetsequence at their 3′ ends, and a T7 promoter sequence at their 5′ ends.Primers identified by SEQ ID NOs: 17-22 in Table 2 are promoter-primerscorresponding to the primers identified as SEQ ID NOs: 11-16,respectively. Bases corresponding to T7 promoter sequences in the tableare underlined.

TABLE 2 Polynucleotide Sequences of Amplification Primers Ident- FeatureSequence ifier Target- ATTTCTTGTTCTGTGGTAATCATGTTG SEQ ID complemen- NO:11 tary Target- TTGTTTTTGTAATAGTTGTATTTCTTGTTCTG SEQ ID complemen- NO:12 tary Target- GTTTGTATGTCTGTTGCTATTATGTCTATTAG SEQ ID complemen-TCTTTCTGCTGG NO: 13 tary Target- GTTTGTATGTCTGTTGCTATCATGTTGATTAT SEQ IDcomplemen- TCTTTC NO: 14 tary Target- ATTTGTTTTTGTAATTCTTGTATTTCTATGTCSEQ ID complemen- TGT NO: 15 tary Target- GTTTGTATGTCTGTTGCTATTATGTCTASEQ ID complemen- NO: 16 tary T7 Promo- AATTTAATACGACTCACTATAGGGAGAATTTSEQ ID ter-Primer CTTGTTCTGTGGTAATCATGTTG NO: 17 T7 Promo-AATTTAATACGACTCACTATAGGGAGATTGT SEQ ID ter-PrimerTTTTGTAATAGTTGTATTTCTTGTTCTG NO: 18 T7 Promo-AATTTAATACGACTCACTATAGGGAGAGTTT SEQ ID ter-PrimerGTATGTCTGTTGCTATTATGTCTATTAGTCTT NO: 19 TCTGCTGG T7 Promo-AATTTAATACGACTCACTATAGGGAGAGTTT SEQ ID ter-PrimerGTATGTCTGTTGCTATCATGTTGATTATTCTT NO: 20 TC T7 Promo-AATTTAATACGACTCACTATAGGGAGAATTT SEQ ID ter-PrimerGTTTTTGTAATTCTTGTATTTCTATGTCTGT NO: 21 T7 Promo-AATTTAATACGACTCACTATAGGGAGAGTTT SEQ ID ter-PrimerGTATGTCTGTTGCTATTATGTCTA NO: 22

Preferred sets of primers for amplifying HIV-1, HIV-2, or thecombination of HIV-1 and HIV-2 sequences in the region encoding the p31integrase included a first primer that hybridized the target nucleicacid to be amplified (such as one of the primers listed in Table 2) anda second primer that is complementary to the sequence of an extensionproduct of the first primer (such as one of the primer sequences listedin Table 1). In a highly preferred embodiment, the first primer is apromoter-primer that includes a T7 promoter sequence at its 5′ end.

Preferred Detection Probes

Another aspect of the invention relates to oligonucleotides that can beused as hybridization probes for detecting HIV-1, HIV-2, or thecombination of HIV-1 and HIV-2 nucleic acids. Indeed, methods foramplifying a target sequence present in the nucleic acid of HIV-1 orHIV-2 can include an optional further step for detecting amplicons. Thisprocedure for detecting HIV-1 and/or HIV-2 nucleic acids includes a stepfor contacting a test sample with a hybridization assay probe thathybridizes to the target nucleic acid sequence, or the complementthereof, under stringent hybridization conditions, thereby forming aprobe:target duplex that is stable for detection. Next there is a stepfor determining whether the hybrid is present in the test sample as anindication of the presence or absence of HIV-1 or HIV-2 nucleic acidtarget in the test sample. This may involve detecting the probe:targetduplex, and preferably involves homogeneous assay systems.

Hybridization assay probes useful for detecting HIV-1, HIV-2, or thecombination of HIV-1 and HIV-2 nucleic acids include a sequence of basessubstantially complementary to these target nucleic acid sequences.Thus, probes of the invention hybridize one strand of a target nucleicacid sequence, or the complement thereof. These probes optionally mayhave additional bases outside of the targeted nucleic acid region whichmay or may not be complementary to the target nucleic acid that is to bedetected.

Preferred probes are sufficiently homologous to the target nucleic acidto hybridize under stringent hybridization conditions corresponding toabout 60° C. when the salt concentration is in the range of 0.6-0.9 M.Preferred salts include lithium chloride, but other salts such as sodiumchloride and sodium citrate also can be used in the hybridizationsolution. Example high stringency hybridization conditions arealternatively provided by 0.48 M sodium phosphate buffer, 0.1% sodiumdodecyl sulfate, and 1 mM each of EDTA and EGTA, or by 0.6 M LiCl, 1%lithium lauryl sulfate, 60 mM lithium succinate and 10 mM each of EDTAand EGTA.

Probes in accordance with the invention have sequences substantiallycomplementary to, or substantially corresponding to portions of theHIV-1 and HIV-2 genomes. Certain probes that are preferred for detectingHIV-1, HIV-2, or the combination of HIV-1 and HIV-2 nucleic acidsequences have a probe sequence which includes a target-complementarysequence of bases, and optionally one or more base sequences that arenot complementary to the nucleic acid that is to be detected. Thetarget-complementary sequence of bases preferably is in the length rangeof from 10-100 nucleotides and is able to hybridize to the amplifiednucleic acid. Certain preferred probes that are capable of detectingHIV-1, HIV-2, or the combination of HIV-1 and HIV-2 nucleic acidsequences have target-complementary sequences in the length range offrom 10-100, from 15-60, from 15-45 or from 20-30 nucleotides. Ofcourse, these target-complementary sequences may be linear sequences, ormay be contained in the structure of a molecular beacon, a moleculartorch or other construct having one or more optional nucleic acidsequences that are non-complementary to the target sequence that is tobe detected. As indicated above, probes may be made of DNA, RNA, acombination DNA and RNA, a nucleic acid analog, or contain one or moremodified nucleosides (e.g., a ribonucleoside having a 2′-O-methylsubstitution to the ribofuranosyl moiety).

Certain probes in accordance with the present invention include adetectable label. In one embodiment this label is joined to the probe bymeans of a non-nucleotide linker. For example, detection probes can belabeled with chemiluminescent acridinium ester compounds that areattached via a linker substantially as described in U.S. Pat. No.5,585,481; and in U. S. Pat. No. 5,639,604, particularly as described atcolumn 10, line 6 to column 11, line 3, and in Example 8. Thedisclosures contained in these patent documents are hereby incorporatedby reference.

Table 3 presents the base sequences of some of the hybridization probesthat were used for detecting HIV-1 target sequences, HIV-2 targetsequences, or both HIV-1 and HIV-2 target sequences. Since alternativeprobes for detecting these target nucleic acids can hybridize toopposite-sense strands, the present invention also includesoligonucleotides that are complementary to the sequences presented inthe table.

TABLE 3 Polynucleotide Sequences of Detection Probes Sequence IdentifierCCTGAATTTTAAAAGAAGGGGG SEQ ID NO: 23 CCAGAATTTTAAAAGAAGGGGIGG SEQ ID NO:24 CCACAATTTTAAAAGAAGGGGIGG SEQ ID NO: 25 CCTGAATTTTAAAAGAAGGGGIGG SEQID NO: 26 CATGAATTTTAAAAGAAGGGGA SEQ ID NO: 27 CCTGAATTTTAAAAGAAIGGGGSEQ ID NO: 28 CCIGAATTTTAAAAGAAGGGGG SEQ ID NO: 29CCIIAATTTTAAAAGAAGGGGG SEQ ID NO: 30 AAAGAAIGGIGGGGATIGGGIGG SEQ ID NO:31 AAAGAAIGGIGGGGATTGGGIGG SEQ ID NO: 32 AATTTTAAAAGAAGAGGIGGGATTGGGGGSEQ ID NO: 33 CAATTTTAAAAGAAGGGGIGGG SEQ ID NO: 34GAATTTTAAAAGAAGIGGGGIG SEQ ID NO: 35 GAAUUUUAAAAGAAGGGGIGGG SEQ ID NO:36

As indicated above, any number of different backbone structures can beused as a scaffold for the base sequences of the invented hybridizationprobes. In certain highly preferred embodiments, the probe includes amethoxy backbone, or at least one methoxy linkage in the nucleic acidbackbone.

Selection and Use of Capture Oligonucleotides

Preferred capture oligonucleotides include a first sequence that issubstantially complementary to a target sequence (i.e., a“target-complementary” sequence) covalently attached to a secondsequence (i.e., a “tail” sequence) that serves as a target forimmobilization on a solid support. Any backbone to link the basesequence of a capture oligonucleotide may be used. In certain preferredembodiments the capture oligonucleotide includes at least one methoxylinkage in the backbone. The tail sequence, which is preferably at the3′ end of a capture oligonucieotide, is used to hybridize to acomplementary base sequence to provide a means for capturing thehybridized target nucleic acid in preference to other components in thebiological sample.

Although any base sequence that hybridizes to a complementary basesequence may be used in the tail sequence, it is preferred that thehybridizing sequence span a length of about 5-50 nucleotide residues.Particularly preferred tail sequences are substantially homopolymeric,containing about 10 to about 40 nucleotide residues, or more preferablyabout 14 to about 30 residues. A capture oligonucleotide according tothe present invention may include a first sequence that hybridizes to atarget polynucleotide, and a second sequence that hybridizes to anoligo(dT) stretch immobilized to a solid support.

Using the components illustrated in FIG. 1, one assay for detectingHIV-1, HIV-2, or the combination of HIV-1 and HIV-2 sequences in abiological sample includes the steps of capturing the target nucleicacid using the capture oligonucleotide, amplifying the captured targetregion using at least two primers, and detecting the amplified nucleicacid by first hybridizing a labeled probe to a sequence contained in theamplified nucleic acid, and then detecting a signal resulting from thebound labeled probe.

The capturing step preferably uses a capture oligonucleotide where,under hybridizing conditions, one portion of the capture oligonucleotidespecifically hybridizes to a sequence in the target nucleic acid and atail portion serves as one component of a binding pair, such as a ligand(e.g., a biotin-avidin binding pair) that allows the target region to beseparated from other components of the sample. Preferably, the tailportion of the capture oligonucleotide is a sequence that hybridizes toa complementary sequence immobilized to a solid support particle.Preferably, first, the capture oligonucleotide and the target nucleicacid are in solution to take advantage of solution phase hybridizationkinetics. Hybridization produces a capture oligonucleotide:targetnucleic acid complex which can bind an immobilized probe throughhybridization of the tail portion of the capture oligonucleotide with acomplementary immobilized sequence. Thus, a complex comprising a targetnucleic acid, capture oligonucleotide and immobilized probe is formedunder hybridization conditions. Preferably, the immobilized probe is arepetitious sequence, and more preferably a homopolymeric sequence(e.g., poly-A, poly-T, poly-C or poly-G), which is complementary to thetail sequence and attached to a solid support. For example, if the tailportion of the capture oligonucleotide contains a poly-A sequence, thenthe immobilized probe would contain a poly-T sequence, although anycombination of complementary sequences may be used. The captureoligonucleotide may also contain “spacer” residues, which are one ormore bases located between the base sequence that hybridizes to thetarget and the base sequence of the tail that hybridizes to theimmobilized probe. Any solid support may be used for binding the targetnucleic acid:capture oligonucleotide complex. Useful supports may beeither matrices or particles free in solution (e.g., nitrocellulose,nylon, glass, polyacrylate, mixed polymers, polystyrene, silanepolypropylene and, preferably, magnetically attractable particles).Methods of attaching an immobilized probe to the solid support are wellknown. The support is preferably a particle which can be retrieved fromsolution using standard methods (e.g., centrifugation, magneticattraction of magnetic particles, and the like). Preferred supports areparamagnetic monodisperse particles (i.e., uniform in size±about 5%).

Retrieving the target nucleic acid:capture oligonucleotide:immobilizedprobe complex effectively concentrates the target nucleic acid (relativeto its concentration in the biological sample) and purifies the targetnucleic acid from amplification inhibitors which may be present in thebiological sample. The captured target nucleic acid may be washed one ormore times, further purifying the target, for example, by resuspendingthe particles with the attached target nucleic acid:captureoligonucleotide:immobilized probe complex in a washing solution and thenretrieving the particles with the attached complex from the washingsolution as described above. In a preferred embodiment, the capturingstep takes place by sequentially hybridizing the capture oligonucleotidewith the target nucleic acid and then adjusting the hybridizationconditions to allow hybridization of the tail portion of the captureoligonucleotide with an immobilized complementary sequence (e.g., asdescribed in PCT No. WO 98/50583). After the capturing step and anyoptional washing steps have been completed, the target nucleic acid canthen be amplified. To limit the number of handling steps, the targetnucleic acid optionally can be amplified without releasing it from thecapture oligonucleotide.

Useful capture oligonucleotides may also contain mismatches to thesequence of the nucleic acid molecule that is to be amplified.Successful target capture and nucleic acid amplification can be achievedas long as the mismatched sequences hybridize to the nucleic acidmolecule containing the sequence that is to be amplified. Indeed,oligonucleotides for the capture of HIV-1 nucleic acids, as described inthe published international patent application identified by WO03/106714, were used to practice the methods disclosed herein, includingmethods of detecting HIV-2.

Preferred Methods for Amplifying and Detecting Target PolynucleotideSequences

Preferred methods of the present invention are described and illustratedby the Examples presented below. FIG. 1 schematically illustrates onesystem that may be used for detecting a target region of the viralgenome (shown by a thick solid horizontal line). This basic systemincludes four oligonucleotides (shown by the shorter solid lines): onecapture oligonucleotide that includes a sequence that hybridizes to asequence in the target region and a tail (“T”) that hybridizes to acomplementary sequence immobilized on a solid support to capture thetarget region present in a biological sample; one T7 promoter-primerwhich includes a sequence that hybridizes specifically to an HIV-1 orHIV-2 sequence in the target region and a T7 promoter sequence (“P”)which, when double-stranded, serves as a functional promoter for T7 RNApolymerase; one non-T7 primer which includes a sequence that hybridizesspecifically to a first strand cDNA made from the target region sequenceusing the T7 promoter-primer; and one labeled probe which includes asequence that hybridizes specifically to a portion of the target regionthat is amplified using the two primers.

As indicated above, amplifying the captured target region using the twoprimers can be accomplished by any of a variety of known nucleic acidamplification reactions that will be familiar to those having anordinary level of skill in the art. In a preferred embodiment, atranscription-associated amplification reaction, such as TMA, isemployed. In such an embodiment, many strands of nucleic acid areproduced from a single copy of target nucleic acid, thus permittingdetection of the target by detecting probes that are bound to theamplified sequences. Preferably, transcription-associated amplificationuses two types of primers (one being referred to as a promoter-primerbecause it contains a promoter sequence, labeled “P” in FIG. 1, for anRNA polymerase) two enzymes (a reverse transcriptase and an RNApolymerase), and substrates (deoxyribonucleoside triphosphates,ribonucleoside triphosphates) with appropriate salts and buffers insolution to produce multiple RNA transcripts from a nucleic acidtemplate.

Referring to FIG. 1, during transcription-mediated amplification, thecaptured target nucleic acid is hybridized to a first primer (shown as aT7 promoter-primer). Using reverse transcriptase, a complementary DNAstrand is synthesized from the T7 promoter-primer using the target RNAas a template. A second primer, shown as a non-T7 primer, hybridizes tothe newly synthesized DNA strand and is extended by the action of areverse transcriptase to form a DNA duplex, thereby forming adouble-stranded T7 promoter region. T7 RNA polymerase then generatesmultiple RNA transcripts by using this functional T7 promoter. Theautocatalytic mechanism of TMA employs repetitive hybridization andpolymerization steps following a cDNA synthesis step using the RNAtranscripts as templates to produce additional transcripts, therebyamplifying target region-specific nucleic acid sequences.

The detecting step uses at least one detection probe that bindsspecifically to the amplified RNA transcripts or amplicons describedabove. Preferably, the detection probe is labeled with a label that canbe detected using a homogeneous detection system. For example, thelabeled probe can be labeled with an acridinium ester compound fromwhich a chemiluminescent signal may be produced and detected, asdescribed above. Alternatively, the labeled probe may comprise afluorophore or a paired fluorophore and quencher moiety set. A molecularbeacon is one embodiment of such a labeled probe that may be used in ahomogeneous detection system.

Methods of Detecting HIV-1 and/or HIV-2 Nucleic Acids

Three distinct methods of detecting HIV-1 and HIV-2 nucleic acids inmultiplex assays also have been invented. Each method is distinguishedfrom the other by the use of primers that are cross-reactive oranalyte-specific, and also by the use of probes that are cross-reactiveor analyte-specific.

In the first invented method, independent sets of analyte-specificprimers, meaning a first set of primers specific for HIV-1 nucleic acids(but not HIV-2 nucleic acids) and a second set of primers specific forHIV-2 nucleic acids (but not HIV-1 nucleic acids), are used forsynthesizing amplicons in a single amplification reaction. Thesynthesized amplicons are subsequently detected using a cross-reactiveprobe which is able to detect both HIV-1 amplicons and HIV-2 amplicons.Positive hybridization results obtained using this method indicate thatthe test sample which provided nucleic acid templates for amplificationcontains either HIV-1 or HIV-2, without distinguishing between the twoanalytes.

In the second invented method, a set of cross-reactive primers is usedfor synthesizing HIV-1 amplicons and/or HIV-2 amplicons in a singleamplification reaction. The synthesized amplicons are subsequentlydetected using distinct, analyte-specific probes. One of the probes isspecific for HIV-1 amplicons (but not HIV-2 amplicons) while another ofthe probes is specific for HIV-2 amplicons (but not HIV-1 amplicons).The step for detecting amplicons synthesized using the cross-reactiveprimers can involve either combining the analyte-specific probes in asingle hybridization reaction, or separately hybridizing each of theanalyte-specific probes with aliquots containing the products of theamplification reaction. If the probes are combined in a singlehybridization reaction, then a positive result indicates that a testsample contains either HIV-1 or HIV-2, without distinguishing betweenthe two analytes. Alternatively, if the probes are separately hybridizedwith independent aliquots of the amplification reaction, then a positiveresult in one of the hybridization reactions will indicate that theanalyte complementary to the probe contained in the reaction was presentin the test sample that provided nucleic acid templates foramplification.

In the third invented method, a set of cross-reactive primers is usedfor synthesizing HIV-1 amplicons and/or HIV-2 amplicons in a singleamplification reaction. The synthesized amplicons are subsequentlydetected using a cross-reactive probe which is able to detect both HIV-1amplicons and HIV-2 amplicons. Positive hybridization results obtainedusing this method indicate that the test sample which provided nucleicacid templates for amplification contains either HIV-1 or HIV-2, withoutdistinguishing between the two analytes.

The invented cross-reactive primers are particularly useful in multiplexreactions for amplifying HIV-1 and/or HIV-2. Conventional multiplexreactions typically involve the use of a few, or even severalindependent primer sets, with each set of primers being capable ofamplifying a different analyte nucleic acid that may be present in asample undergoing testing. When the number of primers reaches athreshold value, there is the possibility for undesirable primer-primerinteractions to occur. When this is the case, the primers can beconsumed in the production of undesirable extension products, therebyinhibiting the efficient synthesis of analyte-specific amplicons. Asolution to this problem is to use cross-reactive primers that allowamplification of multiple analytes, thereby reducing the number ofprimer species that must be included in the reaction.

Another benefit of the invented cross-reactive primers and probes alsorelates to multiplex amplification reactions. More particularly, thepreferred use of at least one cross-reactive primer, and more preferablyat least one set of two cross-reactive primers, in a multiplexamplification reaction affords redundancy in the detection of at leastone of the subject analytes. This redundant detection is highlyadvantageous when one of the analytes is prone to mutation or exists inalternative forms that could be missed by the use of a single set ofamplification primers. For example, if a multiplex amplificationreaction is a capable of detecting HIV-1 and HIV-2, it is desirable, inaccordance with the present invention, to carry out amplificationreactions using at least one set of primers that are capable ofamplifying both HIV-1 and HIV-2. When this is the case, thecross-reactive primers will provide a redundant means for amplifying theHIV-1 analyte polynucleotide. Similarly, a cross-reacting probe capableof hybridizing HIV-1 amplicons and HIV-2 amplicons provides a redundantmeans for detecting HIV-1 analyte polynucleotides in a hybridizationreaction that contains a probe specific for HIV-1 and not HIV-2.

Notably, the desired level of cross-reactivity among the primers ofmultiplex assays capable of amplifying portions of more than two analytepolynucleotides is limited. For example, when a multiplex reaction iscapable of amplifying portions of three different analytepolynucleotides, a set of cross-reacting primers in accordance with theinvention should be capable of amplifying portions of only two of thethree analytes. When a multiplex reaction is capable of amplifyingportions of four different analyte polynucleotides, a set ofcross-reacting primers in accordance with the invention should becapable of amplifying portions of either only two of the four analytesor only three of the four analytes. Generally speaking, a set ofcross-reacting primers in accordance with the invention should becapable of amplifying portions of fewer than the total number of analytepolynucleotides that can be amplified in the multiplex reaction. Thisclearly is distinct from a situation in which all polynucleotideanalytes of a multiplex reaction are amplified, as may be the case whenone of the primers in a reaction is an oligo dT primer.

Kits for Detecting HIV-1 HIV-2, or the Combination of HIV-1 and HIV-2Nucleic Acids

The present invention also embraces kits for performing polynucleotideamplification reactions using viral nucleic acid templates. Certainpreferred kits include a hybridization assay probe that has atarget-complementary sequence of bases, and optionally include primersor other ancilary oligonucleotides for amplifying the target that is tobe detected by the hybridization assay probe. Other preferred kitscontain a pair of oligonucleotide primers that may be used foramplifying target nucleic acids in an in vitro amplification reaction.Exemplary kits include first and second amplification oligonucleotidesor primers that are complementary to opposite strands of a targetnucleic acid sequence that is to be amplified. The kits may furthercontain one or more probes for detecting the amplification productssynthesized by the action of the primers which are contained in the kit.Still other kits in accordance with the invention may additionallyinclude capture oligonucleotides for purifying template nucleic acidsaway from other species prior to amplification.

The general principles of the present invention may be more fullyappreciated by reference to the following non-limiting Examples.

Example 1 describes procedures that identified some of the hybridizationprobes which subsequently were used in assays for detecting HIV-1,HIV-2, or the combination of HIV-1 and HIV-2 nucleic acids. Moreparticularly, the following procedures employed syntheticoligonucleotides as targets for hybridization probes. As indicatedbelow, one of the probes tested in the procedure exhibited substantiallyequivalent specificity for HIV-1 and HIV-2 targets.

EXAMPLE 1 Oligonucleotide Probes for Detecting HIV-1 and/or HIV-2

Synthetic target oligonucleotides were prepared according to standardlaboratory procedures using 2′-OMe nucleotide analogs to mimic RNAstructures. The model HIV-1 target had the sequence ofTCCCCCCTTTTCTTTTAAAATTGTGGATGA (SEQ ID NO:37), while the model HIV-2target had the sequence of TTCCTCCCCTTCTTTTAAAATTCATGCAAT (SEQ IDNO:38). Probes for hybridizing these synthetic targets had the sequencesgiven in Table 3, and were also prepared using 2′-OMe nucleotideanalogs.

Hybridization reactions included about 2×10⁶ RLUs of AE-labeled probehaving a specific activity of about 1-7×10⁸ RLU/pmole, and about 0.5pmoles of synthetic target oligonucleotide. Negative control reactionsomitted the target oligonucleotide. The probes listed in Table 3 wereeach labeled with an AE moiety joined to the oligonucleotide structureby an internally disposed non-nucleotide linker according to proceduresdescribed in U.S. Pat. Nos. 5,585,481 and 5,639,604, the disclosures ofthese patents having been incorporated by reference hereinabove. Thelinkers on the probes of SEQ ID NO:23, SEQ ID NO:30, SEQ ID NO:27, SEQID NO:28 and SEQ ID NO:29 were located between positions 7 and 8. Thelinkers on the probes of SEQ ID NO:24 and SEQ ID NO:25 were locatedbetween positions 13 and 14. The linker on the probe of SEQ ID NO:26 waslocated between positions 12 and 13. The linkers on the probes of SEQ IDNO:31 and SEQ ID NO:32 were located between positions 17 and 18. Thelinker on the probe of SEQ ID NO:33 was located between positions 26 and27. The linker on the probe of SEQ ID NO:34 was located betweenpositions 9 and 10. The linker on the probe of SEQ ID NO:35 was locatedbetween positions 8 and 9. The linker on the probe of SEQ ID NO:36 waslocated between positions 11 and 12. Use of all of these differentlinker positions confirmed the versatility of this labeling technique.Probe hybridizations were carried out at 60° C. for 15 minutes in 50 μlvolumes of a Tris-buffered solution that included the reagents used inthe amplification reaction described in Example 2. Hybridizationreactions were followed by addition of an aliquot of 0.15 M sodiumtetraborate (pH 8.5), and 1% TRITON X-100 (Union Carbide Corporation;Danbury, Conn.). These mixtures were first incubated at 60° C. for 10minutes to inactivate the chemiluminescent label joined to unhybridizedprobe, and cooled briefly to 4° C. prior to reading the hybridizationsignal. Chemiluminescence due to hybridized probe in each sample wasassayed using a LUMISTAR GALAXY luminescence microplate reader (BMGLabtechnologies Inc.; Durham, N.C.) configured for automatic injectionof 1 mM nitric acid and 0.1% (v/v) hydrogen peroxide, followed byinjection of a solution containing 1 N sodium hydroxide. Results for thechemiluminescent reactions were measured in relative light units (RLU).Representative results from this procedure are summarized in Table 4 foreach of the three different target regions. In this procedure, thesignal/noise value corresponded to the chemiluminescent signal (measuredin RLU) generated by label associated with specifically hybridized probedivided by a background signal measured in the absence of a targetnucleic acid. Each value represents the average of 5 replicates.

TABLE 4 Probe Hybridization Results HIV-1 Target HIV-2 Target (SEQ IDNO: 37) (SEQ ID NO: 38) RLU RLU remaining remaining p31 Integrase Regionas % of T₀ Signal/ as % of T₀ Signal/ Probe value Noise value Noise SEQID NO: 23 2 1 101 58 SEQ ID NO: 24 7 1 61 8 SEQ ID NO: 25 110 12 107 11SEQ ID NO: 26 6 1 72 13 SEQ ID NO: 27 7 1 70 11 SEQ ID NO: 28 1 1 49 35SEQ ID NO: 29 3 1 57 18 SEQ ID NO: 30 4 1 30 10 SEQ ID NO: 31 80 7 42 4SEQ ID NO: 32 60 10 14 2 SEQ ID NO: 33 50 34 2 1 SEQ ID NO: 34 21 3 50 8SEQ ID NO: 35 2 1 54 31 SEQ ID NO: 36 13 1 61 5

The results presented in Table 4 showed that some of the probes testedin the procedure gave a strong hybridization signal followinginteraction with the one or both of the target sequences. Only some ofthe probes used in the procedure gave S/N values substantially greaterthan 10 when hybridized with at least one of the synthetic targets.

Interestingly, very subtle differences distinguished useful probesequences from each other. For example, when compared with the probehaving the sequence of SEQ ID NO:24, the probe of SEQ ID NO:26 differedby only two out of twenty-four nucleotide positions and retained astrong specificity for the HIV-2 target. On the other hand, a probehaving the sequence of SEQ ID NO:25 differed from the probe of SEQ IDNO:24 by only one of these two different nucleotide positions and didnot exhibit specificity for the HIV-2 target. Indeed, the probe of SEQID NO:25 failed to exhibit substantial specificity for either of the twotargets and was found to be capable of hybridizing with substantiallyequal specificity to the HIV-1 and HIV-2 targets. In all three cases,the probes included a single inosine base analog and so did notcorrespond to any naturally occurring HIV-1 or HIV-2 nucleic acidsequence.

The unusual hybridization properties of the probe having the sequence ofSEQ ID NO:25 rendered it highly useful for detecting either HIV-1 orHIV-2. A positive result indicating hybridization of this probe to theproducts of a multiplex reaction that is capable of amplifying eitherHIV-1 or HIV-2 indicates that the test sample which provided the nucleicacid templates for amplification contained at least one of the twoanalytes. Use of the cross-reactive probe of SEQ ID NO:25 as a componentin a hybridization probe reagent containing a separate probe specificfor HIV-1 (but not HIV-2) advantageously provides a means forredundantly detecting the HIV-1 analyte while simultaneously providing ameans for detecting an HIV-2 analyte. Although somewhat less preferredbecause of reduced signal recovery (see Table 4), a probe having thesequence of SEQ ID NO:31 can be used in place of the probe of SEQ IDNO:25 in applications where it is desirable to employ a probe that isable to hybridize with HIV-1 and HIV-2 nucleic acids.

Highly preferred embodiments of the invention employ the cross-reactiveprobe of SEQ ID NO:25 for detection of either HIV-1 or HIV-2 nucleicacids. For example, kits can include in packaged combination: the probeof SEQ ID NO:25 and a set of oligonucleotide primers that are specificfor HIV-1 (but not HIV-2) and a set of oligonucleotide primers that arespecific for HIV-2 (but not HIV-1). An alternative kit can include inpackaged combination: the probe of SEQ ID NO:25 and a set ofoligonucleotide primers that are cross-reactive with HIV-1 and HIV-2,meaning that they are capable of amplifying both HIV-1 and HIV-2 nucleicacids.

Probes that were useful for detecting HIV-2 nucleic acids, but not HIV-1nucleic acids, included: SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:35, andSEQ ID NO:36.

Preferred primer combinations for amplifying HIV-1, HIV-2, or thecombination of HIV-1 and HIV-2 nucleic acids were identified in a seriesof procedures that employed virions as the source of nucleic acidtemplates. Promoter-primers and opposite strand primers were screened incombination using the method described below. Although these procedureswere particularly carried out using a Transcription MediatedAmplification (TMA) protocol, the primers disclosed herein may be usedto produce amplicons by alternative in vitro nucleic acid amplificationmethods that will be familiar to those having an ordinary level of skillin the art.

Example 2 describes methods that identified primers useful foramplifying the p31 integrase region of HIV-1 and/or HIV-2.

EXAMPLE 2 Identification of Amplification Primers

A high-titer cell lysate containing HIV-2 B6 virus particles served asthe source of HIV-2 template sequences in amplification reactions thatemployed paired sets of primers. Virus-negative serum was used toprepare diluted stocks containing either 100 copies/ml of the HIV-1nucleic acid template, or 300 copies/ml of the HIV-2 nucleic acidtemplate. In a single instance, a stock containing 100 copies/ml of theHIV-2 nucleic acid template was prepared. Nucleic acids underwentspecimen processing and target capture prior to amplificationessentially according to the procedures disclosed in publishedInternational Patent Application No. PCT/US2000/18685, except thattemplates were captured using oligonucleotides described in thepublished international patent application identified by WO 03/106714for the capture of HIV-1 nucleic acids. Notably, captureoligonucleotides do not participate in the amplification or detectionsteps of the assay. Virus-containing samples having volumes of 0.5 mlwere combined with a target-capture reagent to facilitate nucleic acidrelease and hybridization to capture oligonucleotides disposed onmagnetic beads. TMA reactions were carried out essentially as describedby Kacian et al., in U.S. Pat. No. 5,399,491, the disclosure of thisU.S. patent having been incorporated by reference hereinabove.Promoter-primers included a T7 promoter sequenceAATTTAATACGACTCACTATAGGGAGA (SEQ ID NO:39) upstream of atarget-complementary sequence. Amplification reactions were conductedfor various primer combinations using 15 pmoles of each primer in 100 μlof reaction buffer. Isolated target nucleic acids were combined withprimers in a standard nucleic acid amplification buffer, heated to 60°C. for 10 minutes and then cooled to 42° C. to facilitate primerannealing. Moloney Murine Leukemia Virus (MMLV) reverse transcriptase(5,600 units/reaction) and T7 RNA polymerase (3,500 units/reaction) werethen added to the mixtures. Amplification reactions were carried out ina Tris-buffered solution (pH 8.2 to 8.5) containing KCl,deoxyribonucleoside 5′-triphosphates, ribonucleoside 5′-triphosphates,N-Acetyl-L-Cysteine, and 5% (w/v) glycerol, as will be familiar to thosehaving an ordinary level of skill in the art. After a one hourincubation at 42° C., the entire 100 μl amplification reaction wassubjected to a hybridization assay essentially as described in Example 1using an independent HIV-1 specific probe which did not cross-hybridizewith HIV-2, and the HIV-2 specific probe of SEQ ID NO:23 which did notcross-hybridize with HIV-1. The probes were labeled with acridiniumester to specific activities of about 1-7×10⁸ RLU/pmol and then used inamounts equivalent to about 2×10⁶ RLU for each hybridization reaction.Specifically hybridized probe was quantified following chemicalinactivation of the chemiluminescent label associated withnon-hybridized probe in a homogeneous assay essentially as described inExample 1. Trials were conducted using replicates of 10. To be judged asa positive result, the chemiluminescent signal indicating probehybridization must have exceeded 50,000 RLU in an assay.

Table 5 presents results from amplification procedures that wereconducted using different combinations of amplification primers.Numerical values appearing in the table represent the percentage ofpositive trials.

TABLE 5 Amplification of HIV-1 and HIV-2 Polynuclcotide Sequences UsingVarious Primer Combinations T7 Primer Target-Complementary Sequencenon-T7 primer Target (c/ml) SEQ ID NO: 11 SEQ ID NO: 12 SQ ID NO: 13 SEQID NO: 14 SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 1 HIV1 (100)  0%  0%100% ND ND 100% HIV2 (100) 100% 100%  0% ND ND  0% SEQ ID NO: 2 HIV1(100)  0%  0% 100% 100% 100% 100% HIV2 (100) 100% 100% 100% 100% 100% NDHIV2 (300) ND ND ND ND ND 100% SEQ ID NO: 3 HIV1 (100)  0% ND ND ND NDND HIV2 (100) 100% 100% 100% ND ND ND SEQ ID NO: 4 HIV1 (100) ND ND ND100% ND ND HIV2 (300) ND ND ND  0% ND ND SEQ ID NO: 5 HIV1 (100) ND NDND 100% ND ND HIV2 (300) ND ND ND  0% ND ND SEQ ID NO: 6 HIV1 (100) NDND ND 100% ND ND HIV2 (300) ND ND ND  0% ND ND SEQ ID NO: 7 HIV1 (100)ND ND ND 100% ND ND HIV2 (300) ND ND ND 100% ND ND SEQ ID NO: 8 HIV1(100) ND ND ND 100% ND ND HIV2 (300) ND ND ND  57% ND ND SEQ ID NO: 9HIV1 (100) ND ND ND  80% ND ND HIV2 (300) ND ND ND  50% ND ND “ND”indicates “not done” (primer pair not tested)

The results presented in Table 5 showed that some of the primercombinations gave very high levels of HIV-1 and HIV-2 detectability,even at levels as low as 50 copies of the viral template per reaction.More specifically, excellent results were obtained using a primer havingthe target-complementary sequence of SEQ ID NO:2 in combination with aprimer having the target-complementary sequence of any of SEQ ID NO:13,SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16. Excellent results also wereachieved using a primer having the target-complementary sequence of SEQID NO:14 in combination with a primer having the target-complementarysequence of any of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. Thetarget-complementary portions of these latter three primers allconformed to the consensus sequence TAGAGACAGNAGTACNAATGGC (SEQ IDNO:40), where position 10 is occupied by C, T or I, and where position16 is occupied by T or I. Primers conforming to this consensus arepreferred for amplifying HIV-1 or HIV-2 target nucleic acids. Acombination of primers having the target-complementary sequences of SEQID NO:14 and SEQ ID NO:7 advantageously is capable of amplifying thelargest number of genetic variants of HIV-1 and HIV-2. Notably, nofalse-positive reactions were observed in these procedures.

Although the foregoing Example describes assays conducted usingindependent probes that were specific for HIV-1 (but not for HIV-2) andHIV-2 (but not for HIV-1), the invention also embraces compositions,kits and methods employing probes that are cross-reactive for HIV-1 andHIV-2. Particular examples of cross-reactive probes that can be used inconjunction with any of the above-described primer combinations have thesequences of SEQ ID NO:25 and SEQ ID NO:31.

The ability of a selected set of oligonucleotides to amplify and detecta variety of different HIV-2 isolates was next demonstrated.

Example 3 describes procedures that demonstrated the invented primersand probe were useful for detecting a broad range of HIV-2 isolates.

EXAMPLE 3 Broad Range of Detectability for HIV-2

Primers having the target-complementary sequences of SEQ ID NO:7 and SEQID NO. 14 were used in combination for amplifying HIV-2 template nucleicacids from seven different HIV-2 strains that were available ashigh-titer lysates. These specimens were diluted in virus-negative serumto produce stocks having viral template concentrations of 300 copies/ml.As in the previous Example, virus-containing samples having volumes of0.5 ml were combined with a target-capture reagent to facilitate nucleicacid release and hybridization to capture oligonucleotides that weredisposed on magnetic beads. Amplification reactions were carried out asdescribed in the proceeding Example. Amplicons were detected essentiallyas described in Example 1, except that an AE-labeled, HIV-2 specificprobe having the sequence of SEQ ID NO:23 was used, and the detectionstep was carried out using a LEADER HC+luminometer (Gen-ProbeIncorporated, CA). Assays yielding specific hybridization signals of atleast 50,000 RLUs were judged as being positive. All assays were carriedout in replicates of ten. Results from these procedures are presented inTable 6.

TABLE 6 Amplification and Detection of Different HIV-2 Isolates HIV-2Strain % Positive (N = 10) HIV-2 B2 100 HIV-2 B3 100 HIV-2 B4 100 HIV-2B5 100 HIV-2 B7 100 HIV-2 B8 100 HIV-2 B9 100

The results presented in Table 6 showed that the cross-reactive primershaving the target-complementary sequences of SEQ ID NO:7 and SEQ IDNO:14 were capable of amplifying nucleic acids from a variety ofdifferent strains of HIV-2, and similarly that an HIV-2 specific probehaving the sequence of SEQ ID NO:23 was capable of detecting nucleicacids from a variety of different HIV-2 strains. These primers and thisprobe represent preferred embodiments of the invention.

Although the foregoing Example illustrated an assay based on thecombined use of cross-reactive primers and an analyte-specific probe,the present invetion also embraces embodiments wherein cross-reactiveprimers are used in combination, or packaged in a kit, with a probe thatalso is cross-reactive, meaning that the probe is capable ofindependently hybridizing to HIV-1 and HIV-2 nucleic acids or amplicons.Particular examples of cross-reactive primers and cross-reactive probesare disclosed herein. For instance, the illustrative HIV-2 specificprobe used in this Example could have been substituted by one of thecross-reactive probes identified by SEQ ID NO:25 or SEQ ID NO:31.

The following Example demonstrates that two different primercombinations were capable of amplifying an HIV-2 template nucleic acidat an amount equal to 150 copies/reaction. Significantly, theseamplification and detection procedures were performed in a reactionmixture that was capable of multiplex amplification of HIV-1, HIV-2, HBVand HCV. These results showed that the presence of primers specific forextraneous targets did not adversely impact detection of HIV-2.

Example 4 describes procedures that demonstrated how the inventedprimers could be combined in a multiplex nucleic acid amplificationreaction capable of detecting HIV-1, HIV-2, HBV and HCV.

EXAMPLE 4 Amplification of HIV-2 Nucleic Acids in a Multiplex Assay

Primers having the sequences of SEQ ID NO:20 (target-complementarysequence of SEQ ID NO:14) and either SEQ ID NO:2 or SEQ ID NO:7 wereadded to a reaction formulation that included primers capable ofamplifying analytes that included HIV-1, HBV and HCV. Multiplex assayformulations for performing target capture, amplification andprobe-based detection of these targets are disclosed in the publishedinternational patent application identified by WO 03/106714, thedisclosure of which is incorporated by reference. As in Example 2,samples containing HIV-1 virions or HIV-2 virions were prepared bydiluting high-titer stocks with virus-negative serum. Target capture andnucleic acid amplification reactions were performed using 0.5 ml ofdiluted virus sample, as described herein. Detection of HIV-2 ampliconsby the procedures described above was carried out using the HIV-2specific probe of SEQ ID NO:23, together with probes specific fordetecting HIV-1, HBV and HCV amplicons. Assays yielding specifichybridization signals of at least 50,000 RLUs were judged as beingpositive. Results from these procedures appear in Table 7.

TABLE 7 Detection of HIV-2 in a Multiplex Assay Target-ComplementaryPrimer Sequences Target % Positive SEQ ID NO: 2 HIV-1 Type B (100 c/ml)100% (N = 10) SEQ ID NO: 14 HIV-2 B8 (300 c/ml) 100% (N = 10) SEQ ID NO:7 HIV-1 Type B (100 c/ml) 100% (N = 40) SEQ ID NO: 14 HIV-2 B7 (300c/ml) 100% (N = 40)

The results presented in Table 7 confirmed that different combinationsof the invented primers were capable of efficiently detecting HIV-2target nucleic acids in multiplex reactions that were also capable ofamplifying and detecting HIV-1, HBV and HCV.

In addition to the above-described assay which detects HIV-1 and HIV-2sequences in the regions encoding the p31 integrase, a second targetregion, located within the gene encoding the p51 reverse transcriptase(RT), was also found to be useful for detecting HIV-1 and HIV-2 nucleicacids. Methods used to make this second demonstration were essentiallyas described above. Oligonucleotide probes used in the procedures foridentifying cross-reactive probes in the p51 RT target region had thesequences presented in the Table 8.

TABLE 8 Sequences of Detection Probes Sequence IdentifierAGGCAGUAUACUGCAUUUACCIUACC SEQ ID NO: 41 GTATACTGCATTTACCCTACC SEQ IDNO: 42 AGGAAGUAUACUGCAUUUACCIUACC SEQ ID NO: 43AGGAAGUAUACUGCAUUUACCAUACC SEQ ID NO: 44

Example 5 describes procedures used to identify candidate cross-reactiveprobes that hybridized to the p51 RT regions of HIV-1 and HIV-2 nucleicacids.

EXAMPLE 5 Oligonucleotide Probes for Detecting HIV-1 and/or HIV-2

Synthetic target oligonucleotides were prepared according to standardlaboratory procedures. The model HIV-1 target had the sequence ofCUAGGUAUGGUAAAUGCAGUAUACUUC (SEQ ID NO:45), while the model HIV-2 targethad the sequence of GAUGGUAGGGUAAAUGCAGUAUACU (SEQ ID NO:46). The HIV-1and HIV-2 targets were both synthesized using RNA precursors. Probes forhybridizing these synthetic targets had the sequences given in Table 8,and were prepared using 2′-OMe nucleotide analogs.

Hybridization reactions included about 2×10⁶ RLUs of AE-labeled probehaving a specific activity of about 1-7×10⁸ RLU/pmole, and about 0.5pmoles of synthetic target oligonucleotide. Negative control reactionsomitted the target oligonucleotide. The probes listed in Table 8 wereeach labeled with an AE moiety joined to the oligonucleotide structureby an internally disposed non-nucleotide linker according to proceduresdescribed in U.S. Pat. Nos. 5,585,481 and 5,639,604, the disclosures ofthese patents having been incorporated by reference hereinabove. Thelinker on the probe of SEQ ID NO:42 alternatively was located betweennucleotides 7 and 8, between nucleotides 11 and 12, or betweennucleotides 12 and 13. The linkers on the probes of SEQ ID NO:41, SEQ IDNO:43, and SEQ ID NO:44 were all located between nucleotides 12 and 13.Use of these different linker positions confirmed the versatility ofthis labeling technique. Probe hybridizations were carried out at 60° C.for 15 minutes in 50 μl volumes of a Tris-buffered solution thatincluded the reagents used in the amplification reaction described inExample 2. Hybridization reactions were followed by addition of analiquot of 0.15 M sodium tetraborate (pH 8.5), and 1% TRITON X-100(Union Carbide Corporation; Danbury, Conn.). These mixtures were firstincubated at 60° C. for 10 minutes to inactivate the chemiluminescentlabel joined to unhybridized probe, and cooled briefly to 4° C. prior toreading the hybridization signal. Chemiluminescence due to hybridizedprobe in each sample was assayed using a LUMISTAR GALAXY luminescencemicroplate reader (BMG Labtechnologies Inc.; Durham, N.C.) configuredfor automatic injection of 1 mM nitric acid and 0.1% (v/v) hydrogenperoxide, followed by injection of a solution containing 1 N sodiumhydroxide. Results for the chemiluminescent reactions were measured inrelative light units (RLU). Representative results from this procedureare summarized in Table 9 for each of the three different probesequences. In this procedure, the signal/noise value corresponded to thechemiluminescent signal (measured in RLU) generated by label associatedwith specifically hybridized probe divided by a background signalmeasured in the absence of a target nucleic acid. Each value representsthe average of 5 replicates.

TABLE 9 Probe Hybridization Results HIV-1 Target HIV-2 Target (SEQ IDNO: 45) (SEQ ID NO: 46) RLU RLU remaining as remaining as p51 RT Region% of T₀ Signal/ % of T₀ Signal/ Probe value Noise value Noise SEQ ID NO:41 66.7 5.1 66.1 5.0 SEQ ID NO: 42 111.4 296.2 101.5 269.8 SEQ ID NO: 43103.7 159.9 97.7 150.7 SEQ ID NO: 44 112.8 162.8 114.1 164.6

The results presented in Table 9 showed that most of the probes testedin the procedure gave strong hybridization signals and signal/noiseratios following interaction with each of the different targetsequences. Notably, the result presented for the probe of SEQ ID NO:42was obtained using the probe having its label positioned betweennucleotides 12 and 13. However, excellent results were also achievedusing the same probe sequence with alternatively positioned labels. Morespecifically, the signal/noise values for the collection of three probesof SEQ ID NO:42 ranged from about 218 up to about 296 for the HIV-1target, and from about 220 to about 282 for the HIV-2 target. Inaddition to probes having the sequence of SEQ ID NO:42, the probes ofSEQ ID NO:43 and SEQ ID NO:44 are also highly preferred for thedetection of either or both of the HIV-1 and HIV-2 target nucleic acids.Of course, the complements of these sequences also are preferredalternatives.

Interestingly, the probes which performed well in the above-describedassay all included target-complementary sequences of 21-26 contiguousbases contained within a consensus sequence given byAGGAAGTATACTGCATTTACCNTACC (SEQ ID NO:62), allowing for RNA and DNAequivalent bases, where “N” is any of A, C or I. The 26-mer probe of SEQID NO:41 did not perform well in the hybridization assay (see Table 9),and does not conform with the consensus. Notably, this poor-performingprobe differed from the cross-reactive probe of SEQ ID NO:43, whichperformed well in the assay, by only a single base change. Thisillustrates the unusual and unexpected nature of the advantageouslycross-reactive probes described above.

Highly preferred embodiments of the invention employ one or more of thecross-reactive probes of SEQ ID NO:42, SEQ ID NO:43 or SEQ ID NO:44 fordetection of either HIV-1 or HIV-2 nucleic acids. However, kits inaccordance with the invention can include in packaged combination: anyprobe having the sequence of SEQ ID NO:42, SEQ ID NO:43 or SEQ ID NO:44and a set of oligonucleotide primers that are specific for HIV-1 (butnot HIV-2) and/or a set of oligonucleotide primers that are specific forHIV-2 (but not HIV-1). An alternative kit can include in packagedcombination: any probe having the sequence of SEQ ID NO:42, SEQ ID NO:43or SEQ ID NO:44 and a set of oligonucleotide primers that arecross-reactive with HIV-1 and HIV-2, meaning primers that are capable ofamplifying both HIV-1 and HIV-2 nucleic acids. The cross-reactive probereagents are particularly preferred for use in methods wherein HIV-1 orHIV-2 amplicons synthesized using the cross-reactive amplificationprimers described in the following Example are detected.

Notably, in certain embodiments it will be desirable to employ probeshaving sequences appended to the 5′ or 3′ ends of thetarget-complementary probe sequences, which sequences are notcomplementary to, meaning that they do not hybridize to, the HIV-1 orHIV-2 amplicons. In these instances it is preferred for the overalllength of the probe molecule to be up to 60, more preferrably up to 26,bases in length. Examples of appended sequences which are notcomplementary to the HIV-1 or HIV-2 amplicons include the “arm”sequences which comprise the “stem” portions of molecular beacons.

Preferred primer combinations for amplifying HIV-1 or HIV-2, or thecombination of HIV-1 and HIV-2 nucleic acids were identified in a seriesof procedures that employed virions as the source of nucleic acidtemplates. Promoter-primers and opposite strand primers were screened incombination using the method described below. Although these procedureswere particularly carried out using a Transcription MediatedAmplification (TMA) protocol, the primers disclosed herein may be usedto produce amplicons by alternative in vitro nucleic acid amplificationmethods that will be familiar to those having an ordinary level of skillin the art. Tables 10 and 11 present the sequences of amplificationprimers that were used in the procedures described under Example 6.Notably, the primers of SEQ ID NOs:51-54 correspond to the primers ofSEQ ID NOs:55-58, respectively, but further include upstream promotersequences that are not complementary to the HIV-1 and HIV-2 targets.

TABLE 10 Sequences of Amplification Primers Sequence IdentifierCTTAGATAAAGAITTCAGGAAGTATA SEQ ID NO: 47 CTTAGATAAAGATTTTAGGAAGTATA SEQID NO: 48 CTTAGATAAAGATTTTAGGCAGTATA SEQ ID NO: 49CTTAGATAAAGATTTTAGGIAGTATA SEQ ID NO: 50

TABLE 11 Sequences of Amplification Primers Identi- Feature Sequencefier Target-com- TTGCTGGTGATCCCTTCCATCCTTGTGG SEQ ID plementary NO: 51Target-com- TTGCTGGTGATCCCTTCCATCCCTGTGG SEQ ID plementary NO: 52Target-com- TTGCTGGTGATCCTTTCCATCC SEQ ID plementary NO: 53 Target-com-TTGCTGGTGATCCCTTCCATCC SEQ ID plementary NO: 54 T7 Promoter-AATTTAATACGACTCACTATAGGGAGA SEQ ID Primer TTGCTGGTGATCCCTTCCATCCTTGTGGNO: 55 T7 Promoter- AATTTAATACGACTCACTATAGGGAGA SEQ ID PrimerTTGCTGGTGATCCCTTCCATCCCTGTGG NO: 56 T7 Promoter-AATTTAATACGACTCACTATAGGGAGA SEQ ID Primer TTGCTGGTGATCCTTTCCATCC NO: 57T7 Promoter- AATTTAATACGACTCACTATAGGGAGA SEQ ID PrimerTTGCTGGTGATCCCTTCCATCC NO: 58

Example 6 describes methods that identified primers useful foramplifying the p51 RT region of HIV-1 and/or HIV-2.

EXAMPLE 6 Identification of Amplification Primers

Tissue culture-derived HIV-2 B6 virus particles served as the source ofHIV-2 template sequences in amplification reactions that employed pairedsets of primers. Virus-negative serum was used to prepare diluted stockscontaining either 100 copies/ml of the HIV-1 nucleic acid template, or300 copies/ml of the HIV-2 nucleic acid template. Nucleic acidsunderwent specimen processing and target capture prior to amplificationessentially according to the procedures disclosed in publishedInternational Patent Application No. PCT/US2000/18685, except thattemplates were captured using oligonucleotides described in thepublished international patent application identified by WO 03/106,714for the capture of HIV-1 nucleic acids. Notably, captureoligonucleotides do not participate in the amplification or detectionsteps of the assay. Virus-containing samples having volumes of 0.5 mlwere combined with a target-capture reagent to facilitate nucleic acidrelease and hybridization to capture oligonucleotides disposed onmagnetic beads. TMA reactions were carried out essentially as describedby Kacian et al., in U.S. Pat. No. 5,399,491, the disclosure of thisU.S. patent having been incorporated by reference hereinabove.Promoter-primers included a T7 promoter sequence given by SEQ ID NO:39upstream of a target-complementary sequence. Amplification reactionswere conducted for various primer combinations using 15 pmoles of eachprimer in 100 μl of reaction buffer. Isolated target nucleic acids werecombined with primers in a standard nucleic acid amplification buffer,heated to 60° C. for 10 minutes and then cooled to 42° C. to facilitateprimer annealing. Moloney Murine Leukemia Virus (MMLV) reversetranscriptase (5,600 units/reaction) and T7 RNA polymerase (3,500units/reaction) were then added to the mixtures. Amplification reactionswere carried out in a Tris-buffered solution (pH 8.2 to 8.5) containingKCl, deoxyribonucleoside 5′-triphosphates, ribonucleoside5′-triphosphates, N-Acetyl-L-Cysteine, and 5% (w/v) glycerol, as will befamiliar to those having an ordinary level of skill in the art. After aone hour incubation at 42° C., the entire 100 μl amplification reactionwas subjected to a hybridization assay essentially as described inExample 1 using a mixture of the above-described probe having thesequence of SEQ ID NO:42. For the purpose of this demonstration, amixture of probes having the sequence of SEQ ID NO:42 with labelspositioned between nucleotides 7 and 8, 11 and 12, and 12 and 13 wereused in a ratio of 2:1:1, respectively. The probes were labeled withacridinium ester to specific activities of about 1-7×10⁸ RLU/pmol andthen used in amounts equivalent to about 2×10⁶ RLU for eachhybridization reaction. Specifically hybridized probe was quantifiedfollowing chemical inactivation of the chemiluminescent label associatedwith non-hybridized probe in a homogeneous assay essentially asdescribed in Example 1. Trials were conducted using replicates of 10. Tobe judged as a positive result, the chemiluminescent signal indicatingprobe hybridization must have exceeded 50,000 RLU in an assay.

Table 12 presents results from amplification procedures that wereconducted using different combinations of amplification primers.Numerical values appearing in the table represent the percentage ofpositive trials.

TABLE 12 Amplification of HIV-1 and HIV-2 Polynucleotide Sequences UsingVarious Primer Combinations T7 Primer Target-Complementary Sequencenon-T7 primer Target (c/ml) SEQ ID NO: 51 SEQ ID NO: 52 SEQ ID NO: 53SEQ ID NO: 54 SEQ ID NO: 47 HIV1 (100) 100% ND 100% ND HIV2 (300) 100%ND 100% ND SEQ ID NO: 48 HIV1 (100) ND 100% ND 100% HIV2 (300) ND 100%ND 100% SEQ ID NO: 49 HIV1 (100) 100% ND 100% ND HIV2 (300) 100% ND 100%ND SEQ ID NO: 50 HIV1 (100) 100% ND 100% ND HIV2 (300) 100% ND 100% ND“ND” indicates “not done” (primer pair not tested)

The results presented in Table 12 showed that all of the selected primercombinations were useful for detecting HIV-1 and HIV-2. Thetarget-complementary portions of useful primers complementary to onestrand of the target to be amplified included a sequence conforming tothe consensus TTGCTGGTGATCCYTTCCATCC (SEQ ID NO:59), where position 14is occupied by C or T, and had a length of up to 28 bases. In apreferred embodiment, the primer conformed to the consensus sequenceTTGCTGGTGATCCYTTCCATCCYTGTGG (SEQ ID NO:60), where positions 14 and 23are independently occupied by C or T. The preferred primers can furtherinclude optional 5′ sequences which are non-complementary to the HIV-1or HIV-2 target to be amplified. The target-complementary portions ofuseful opposite strand primers conformed to the consensus sequenceCTTAGATAAAGANTTYAGGNAGTATA (SEQ ID NO:61), where position 13 is occupiedby T or I, where position 16 is occupied by C or T, and where position20 is occupied by A, C or I. Primers conforming to this consensus arealso preferred for amplifying HIV-1 or HIV-2 target nucleic acids, andcan further include optional 5′ sequences which are non-complementary tothe HIV-1 or HIV-2 target to be amplified. Notably, no false-positivereactions were observed in the procedures described above.

Although the foregoing Example illustrated an assay based on thecombined use of cross-reactive primers and cross-reactive probes, thepresent invention also embraces embodiments wherein cross-reactiveprimers are used in combination, or packaged in a kit, with independent,analyte-specific HIV-1 and HIV-2 probes.

The following Example demonstrates that the cross-reactive p51 RT regionprimers disclosed herein were capable of amplifying HIV-1 and HIV-2templates even when the procedures were performed in a reaction mixturethat was capable of multiplex amplification of HIV-1, HIV-2, HBV andHCV. These results showed that the presence of primers specific forextraneous targets did not adversely impact detection of HIV-2.

Example 7 describes procedures that demonstrated how the inventedprimers could be combined in a multiplex nucleic acid amplificationreaction capable of detecting HIV-1, HIV-2, HBV and HCV.

EXAMPLE 7 Amplification of HIV-2 Nucleic Acids in a Multiplex Assay

Primers having the sequences of SEQ ID NO:57 (target-complementarysequence of SEQ ID NO:53) and SEQ ID NO:49 were added to a reactionformulation that included primers capable of amplifying analytes thatincluded either the combination of HBV and HCV, or the combination ofHIV-1, HBV and HCV. Multiplex assay formulations for performing targetcapture, amplification and probe-based detection of these targets aredisclosed in the published international patent application identifiedby WO 03/106714, the disclosure of which is incorporated by reference.As in Example 2, samples containing HIV-1 virions or HIV-2 virions wereprepared by diluting high-titer stocks with virus-negative serum. Targetcapture and nucleic acid amplification reactions were performed using0.5 ml of diluted virus sample, as described herein. Detection of HIV-2amplicons was carried out using the probe reagent described in thepreceding Example. Detection of HBV and HCV amplicons was carried outusing labeled hybridization probes specific for those targets. Assaysyielding specific hybridization signals of at least 50,000 RLUs werejudged as being positive. Results from these procedures appear in Table13.

TABLE 13 Detection of HIV-2 in a Multiplex Assay Target-ComplementaryPrimer Sequences Target % Positive SEQ ID NO: 53 HIV-1 Type B (100 c/ml)100% SEQ ID NO: 49 HIV-2 Type A (300 c/ml) 100% HCV (60 c/ml) 100% HBV(15 IU/ml) 100%

The results presented in Table 13 confirmed that the invented primerswere capable of efficiently amplifying HIV-1 and HIV-2 target nucleicacids in multiplex reactions that were also capable of amplifying anddetecting other viral targets. Also as shown in the table, low levels ofthe HBV (15 IU/ml) and HCV subtype 2b (60 copies/ml) targets wereefficiently detected in the multiplex reactions capable of amplifyingHIV-1 and HIV-2. Significantly, identical results were obtained usingreaction conditions that included or omitted HIV-1 specificamplification primers. Thus, the disclosed HIV-1/-2 cross-reactiveprimers efficiently detected both HIV-1 and HIV-2, and did not interferewith amplification and detection of the remaining viral targets in themultiplex reaction.

This invention has been described with reference to a number of specificexamples and embodiments thereof. Of course, a number of differentembodiments of the present invention will suggest themselves to thosehaving ordinary skill in the art upon review of the foregoing detaileddescription. Thus, the true scope of the present invention is to bedetermined upon reference to the appended claims.

1. A composition for amplifying both human immunodeficiency virus type 1and 2 (HIV-1 and HIV-2) nucleic acids that may be present in abiological sample, comprising: (a) a first primer consisting of SEQ IDNO:59 and optionally, attached at the 5′ end thereof, a first primerupstream sequence that comprises a restriction endonuclease cleavagesite or an RNA polymerase promoter; and (b) a second primer consistingof SEQ ID NO:61 and optionally, attached at the 5′ end thereof, a secondprimer upstream sequence that comprises a restriction endonucleasecleavage site or an RNA polymerase promoter.
 2. The composition of claim1, wherein said composition does not comprise a pair of HIV-2-specificprimers for amplifying only said HIV-2 nucleic acid and not said HIV-1nucleic acid.
 3. The composition of claim 2, wherein said compositionfurther comprises a pair of HIV-1-specific primers for amplifying onlysaid HIV-1 nucleic acid and not said HIV-2 nucleic acid.
 4. Thecomposition of claim 1, wherein said composition further comprises apair of HIV-1-specific primers for amplifying only said HIV-1 nucleicacid and not said HIV-2 nucleic acid.
 5. The composition of claim 1,wherein said first primer and said second primer are each up to 60 basesin length.
 6. The composition of claim 5, where said first primerconsists of SEQ ID NO:53 and optionally, attached at the 5′ end thereof,said first primer upstream sequence.
 7. The composition of claim 5,where said first primer consists of SEQ ID NO:54 and optionally,attached at the 5′ end thereof, said first primer upstream sequence. 8.The composition of claim 5, wherein the second primer is not attached tosaid second primer upstream sequence.
 9. The composition of claim 8,wherein said second primer is selected from the group consisting of SEQID NO:47, SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50.
 10. Thecomposition of claim 5, wherein the first primer is not attached to saidfirst primer upstream sequence.
 11. The composition of claim 10, whereinthe second primer is not attached to said second primer upstreamsequence.
 12. A method of determining whether a test sample contains atleast one of HIV-1 nucleic acid and HIV-2 nucleic acid, said methodcomprising the steps of: (a) combining said test sample with acomposition according to claim 1 to form a reaction mixture; (b)amplifying, in an in vitro nucleic acid amplification reaction, nucleicacid present in said reaction mixture of step (a); and (c) detecting anHIV-1 amplicon, and HIV-2 amplicon, or a combination of both ampliconssynthesized in step (b), thereby determining that said test samplecontains at least one of HIV-1 nucleic acid and HIV-2 nucleic acid. 13.The method of claim 12, wherein the detecting step comprises detectingin a hybridization reaction.
 14. The method of claim 13, wherein saidhybridization reaction in the detecting step comprises a cross-reactiveprobe which hybridizes independently to said HIV-1 amplicon and to saidHIV-2 amplicon.
 15. The method of claim 14, wherein said hybridizationreaction in the detecting step further comprises an HIV-1 specific probewhich hybridizes only to said HIV-1 amplicon and not to said HIV-2amplicon.
 16. The method of claim 12, wherein said in vitro nucleic acidamplification reaction in the amplifying step is a multiplexamplification reaction that can further amplify, with an assortment oftarget-specific primers, at least one nucleic acid which is differentfrom HIV-1 and HIV-2.
 17. The method of claim 16, wherein said at leastone nucleic acid which is different from HIV-1 and HIV-2 is selectedfrom the group consisting of HBV and HCV.